Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs.

The standard histological processing procedure, which produces excellent staining of sections for most tissues, fails to yield satisfactory results in adult mouse orbits or eyeballs. Here, we show that a protocol using tissue block staining and domestic adhesive tapes resulted in qualified integral serial cryo-sections of whole orbits or eyeballs, and the fine structures were well preserved. The histological processing protocol comprises paraformaldehyde fixation, ethylenediaminetetraacetic acid decalcification, tissue block staining with hematoxylin and eosin, embedding, adhesive tape aided sectioning, and water-soluble mounting. This protocol was proved to be the best in comparison with seven other related existing histological traditional or non-traditional processing methods, according to the staining slice quality. We observed a hundred percent success rate in sectioning, collection, and mounting with this method. The reproducibility tested on qualified section success rates and slice quality scores confirmed that the technique is reliable. The feasibility of the method to detect target molecules in orbits was verified by successful trial tests on block immunostaining and adhesive tape-aided sectioning. Application of this protocol in joints, brains, and so on,-the challenging integral sectioning tissues, also generated high-quality histological staining sections.

1. Introduction

Recent work in visual neuroscience research has concluded that some degenerative eye diseases follow topographically specific retinal ganglion cell death across the retina [1-10] and that the anatomical orientation of the retinae with respect to the orbits is important [11-32]. These studies require visualization of serial whole integrated sections of orbits or eyeballs to determine morphological changes that respond to treatments or genetic modifications.

Whole integrated sectioning of orbits or eyeballs with either traditional paraffin or frozen OCT (optimal cutting temperature) embedding is rather difficult in postnatal mice older than 14 days, especially for relatively thin sections, even with an utmost attempt at precision and perfection [33]. The difficulty in preparing whole integral sections of the orbits or eyeballs arises chiefly because of tremendous differential shrinkage between orbital contents at different temperatures during cutting [34]. These contents of extremely different densities include intraorbital glands, lenses, vitreous humor, the high dense sclera, and the orbital walls of both mineralized and fibril bone tissues [34].

In order to improve current restrictions to secure the whole integral sections of orbital or eyeball, isolation [35-38] of separate tissues or enucleation [39] was performed to study local morphological changes. The technique using a softening lens by chemicals was also used for the sectioning of the eyeball [40]. However, these often lead to a high risk of missing or damaging some parts of tissue and losing orientation of the tissue during histological processing. A comparison between the different treatments is sometimes not valid without unified orientation.

To obtain the whole integral orbital or eyeball sections, an alternative is to use adhesive tape-aided cryo-sectioning. This technique has been well established and successfully applied in histology-sectioning [41]. However, the tape-aided sections can sometimes result in undesirable artifacts such as tears or folds in the steps of staining and dehydrating or clearing, when the adhesive contacts with oil-soluble chemicals and binding forces between the tapes and sections weaken.

In order to circumvent the contacts of the tape section with tape adhesion-diminished oil-soluble reagents during histological processing in the routine staining protocol, we did the orbital or eyeball tissue block staining beforehand, followed by adhesive tape aided sectioning. In the end, the tape-sections were coverslipped with a water-soluble mounting medium, which allows for securing integral and undamaged staining sections. This method was confirmed to be superior compared to 7 other related existing traditional or non-traditional protocols and allows for easy collection of qualified sections with a hundred percent success rate. A trial test on immunostaining with the protocol confirmed the feasibility of this method in detecting target molecules in orbits. Application of the method in other challenging sectioning tissues (e.g., joints, brains, and so on) also produced satisfactory results.

2. Materials and methods

Animal preparation, sample collection, and decalcification

All animal studies (Government of Upper Bavaria; no. 55.2-1-54-2531-25-12), experiments, and procedures were reviewed and approved by the Oberbayern Animal Welfare Committee in Munich, Germany. We confirm that all experiments were performed in accordance with relevant guidelines and regulations.

Female BALB/c mice, with a weight of 18.8–26.4 grams, were delivered from Charles River Laboratory (Sulzfeld, Germany) and allowed to adapt for 1 week before the start of the experiments at the age of 40 weeks. The mice were kept under standard housing conditions.

For orbital preparations, mice were anesthetized and euthanized with isoflurane (CP-Pharma) at a concentration of 1.5–2%. Complete dissection of the orbital and periorbital areas was performed, as described previously [42]. In brief, the heads of the animals were dissected and the skin, the connective tissue around, the brain, and the teeth were removed but left all orbital tissues, eyelids, and adjacent tissues intact. Trimmings were made with coronary cuttings at the positions of Bregma +1.95 and +5.85 mm. Tissue blocks were fixed in 4% paraformaldehyde at 4°C temperature overnight. Decalcification in 15% EDTA (w/v) (ethylenediaminetetraacetic acid) was carried out for 21 days with three times of changing solution, each for one week.

Tissue block HE (hematoxylin and eosin) staining

After post-fixation in Bouin’s (containing 30 mL of saturated picric acid, 10 mL of concentrated formaldehyde, and 2 mL of glacial acetic acid) for 24 h in a hood [buffer formaldehyde (4%, v/v) may serve as an alternate for Bouin’s], the tissue block (~8x6x12 mm3) was subjected to tissue block staining with a modified protocol of the literature [43], and all the tissue blocks during the block staining were processed under ultrasound (Type: RK 100, Bandelin Sonorex, Berlin, Germany). High temperature (e.g., > 60°C) due to ultrasound running should be avoided with counteraction of adding some ice to the water sink. Briefly, the tissues went through 2 h running tap water, dehydration with 80% and absolute ethanol, and clearance with xylol, and were then rehydrated with absolute ethanol and water (repeated for 3 times in each step and 40 min for each time). Tissue block HE staining was started with soaking in Harris’ hematoxylin solution (Hematoxylin Solution, Harris Modified, HS32, Sigma-Aldrich, Germany) for 18 h and were then rinsed in distilled water and 2% acetic acid prepared in 80% ethanol for 2 hrs each to differentiate the tissue, and the tissues were blued with 1% ammonium water and the tap running water for 2 and 3 h respectively. Finally, Eosin counterstaining was performed with 0.5% (w/v) eosin Y (Cat 3137.2, Carl Roth, Germany) prepared in 15% sucrose (w/v) for 18 h.

Embedding, sectioning, and mounting

For the preparation of embedding mold, a slip of a rectangular aluminum foil was rolled onto a 12 mm diameter cylinder and fastened with a piece of adhesive tape. Care was taken to adjust the border of one side of the foil aligned or on a level. A foil tube without a bottom was made after the foil was disengaged from the cylinder (see step-by-step protocol of BTA in Supporting Information in S1 File).

For tissue embedding, the tissue blocks were incubated in 3 changes’ OCT (Optimal cutting temperature compound, VWR Chemicals, Leuven, Belgium) under ultrasound, each for 1 h, and a slide of glass (1x25x75 mm³, #0656.1, Roth, Germany) was placed on a metal block, which had been frozen in dry ice beforehand. One drop of OCT was put on the slide and the oriented orbital tissue block on the OCT immediately after. Special care was taken to embed the optical nerve approximate end side down. The aluminum mold was sheathed onto the specimen and filled with OCT. The frozen tissue block was ready for sectioning (see step-by-step protocol of BTA in Supporting Information in S1 File).

For the selection of adhesive tape, we preferred Tesa 57405 (Tesa, Beiersdorf, Hamburg, Germany) since the adhesive is powerful in low temperature and adhesive tape (two elements-adhesive and plastic film) is colorless and transparent in itself. More importantly, even, the price is very cheap in comparison with commercially available special adhesive tapes, such as Japanese adhesive film (Cryo-film type IIC9, SECTION-LAB, Japan) and it is easy to obtain.

For the adhesive tape-aided sectioning, the tissue block was mounted onto the cryostat chuck using OCT after peeling off the foil mold. The cryostat (CM1850 cryostat; Leica Biosystems, Buffalo Grove, IL) was set at 5 μm section thickness, 5°cutting plane angle, and -20°C chamber temperature. The tissue block was balanced in the chamber for at least 45 min before starting sectioning. A slip of the adhesive tape (15 × 20 mm) was held with fine forceps at a corner of handle, and pressed with the adhesive side of the tape onto the trimmed surface of the sample block. For best adhesion results, light pressure was applied on the tape with soft tissue. It was ensured that the sample cutting surface was entirely covered by the tape, and the cutting blade was sharp. The sample was sectioned slowly, evenly, and continuously without the use of a brush or anti-roll device. A pair of forceps was used to pick up the freshly sectioned tissue by the handle of the tape. The section was then dipped into 20% glycerol two times, placed onto a piece glass of slide with section side up, and finally coverslipped.

Using the microtome, we performed serial coronary sections (5 μm thick, 0.65 mm apart). Cutting was started at the level of Bregma, +1.95 mm, and sections were started to be collected at positions, +2.60, +3.25, +3.90, +4.55, and +5.20 mm (shown in Fig 1) as we did before [42]. The collected sections represented a total distance of 3 to 4 mm and covered the whole eyeball and orbital region of each mouse head.

Fig 1

Images of the orbit or eyeball from a 40 weeks`old BALB/c mouse.

The specimen is processed with the protocol of BTA (block tissue staining, tape-aided sectioning, and aqueous mounting). Serial coronary sections of 5 μm thickness taken at distances of 650 μm are cut at the positions shown in (A). Images from each cutting position are shown in (B). An image of the right orbit magnified in the middle of the serial (Bregma, +3.90 mm) is shown in (C). Images (D-F) are local magnifications of (C) and D1 is the local magnification of (D).

Imaging and acquisition

The macro examination was carried out on the orbital staining sections under an epi-microscope (LED-lupenleuchten, Cat NH99.1, Carl Roth, Germany) and the image was captured with a Canon digital camera (Canon EOS 600D) and recorded with 3456 x2304 pixel resolution.

The HE staining resultant sections were viewed under field illumination and the immune fluorescent staining sections were examined using a filter set (550 nm excitation and 570 nm emission) for Cy-3 and a filter set (365 nm excitation and 450 nm emission) for DAPI, on a Zeiss upright microscope (Carl Zeiss AG, Oberkochen, Germany) using a 2.5X, 10X, and 20X or 40X objective lens (Axioscope, Carl Zeiss). The photos were acquired with an Axiovision digital camera system and recorded with 2560x1920 pixel resolution each.

Comparisons of 8 related protocols

For comparisons with other 7 related existing protocols, 24 mouse heads (a pair of orbits or eyeballs for each head) were employed and processed histologically as shown in Fig 2. Note that the experimental protocol, which was described above in detail is highlighted in bold (see Fig 2).

Fig 2

Flow chart of different protocols.

12 randomly selected heads were subjected to tissue block staining, followed by tape-aided sectioning in 6 heads and without tape-aided sectioning in 6 remaining heads, and then cover-slipping with either water-soluble (20% glycerol in water) or oil-soluble mounting medium DPX (synthetic mounting medium, Sigma) in 3 heads each (refer to Fig 2). The protocol of tissue block staining, tape-aided sectioning, and aqueous mounting was termed as BTA; the protocol of block tissue staining, tape aided sectioning, and oil-soluble mounting as BTO; the protocol of block tissue staining, without tape aided sectioning, and aqueous mounting as BWA; the protocol of block tissue staining, without tape aided sectioning, and oil-soluble mounting as BWO.

The remaining 12 heads were subjected to traditional histological processing,—sectioning with or without adhesive tape aided after tissue block embedding, conventional HE staining of the sections, and finally mounting (refer to Fig 2). The protocol of tape-aided sectioning, staining, and aqueous mounting is termed as TSA; the protocol of tape-aided sectioning, staining, and oil-soluble mounting as TSO; the protocol of without tape-aided sectioning, staining, and aqueous mounting as WSA; the protocol of without tape-aided sectioning, staining, and oil-soluble mounting as WSO.

Each protocol was applied in 3 mouse heads. For comparisons of qualified section yield rates and slice qualities among different related protocols, 10 serial sections were cut at the position of Bregma, +3.90 mm in each animal head. A total of 30 sections were produced for each group of one method. A total of 240 sections (8 methods in total) was subjected to the corresponding treatments mentioned above (Fig 2). The final high-quality staining sections were counted after coverslipping for the determination of the qualified section yield rate. One best quality section of the right-side orbital tissues among the ten for each head was selected for slice quality comparisons among the various protocols according to the criteria of staining section quality.

Determination of qualified section success rate

Some sections were lost during processing. For the unified standard, we took each of the following three facts as one section loss. They included the failure of section mounting, section falling off during processing, or more than half of stained section area damaged or covered with a section fold. The remaining sections (exclusive of section loss) were counted and the qualified section yield rate was defined as the ratio between the quantity of the remaining sections and total sectioning sections (referred to as 30 sections) in each protocol.

Staining section quality evaluation

The total or highest score was 12 for the best quality staining section without any flaws found. A staining section quality score for each section was determined by a deduction of 1 at every one artifact observed from 12. These items of artifacts are listed in Table 1 on three levels of magnification. All sections were evaluated in a blinded fashion (ZL). The average of the scores of the three sections for each protocol stands for the histological quality score of the method.

Table 1

Artifacts observed on a staining section.

Magnification (objective)	Flaws
Macroscopic	section wrinkle or fold
	bubble under section
	tissue fissure
	missing part of tissue
2.5 X	contaminant(e.g., dye dregs) adhere
	shattering
	local tissue dislocation
	distorted tissue structure
10 X	blurring view
	poor staining color contrast
	indistinct tissue structure
	unusual cell or tissue aggregation

Tissue block immunostaining of orbits

The orbital tissues were prepared as described above. For tissue block immunostaining, a one-step method was used to localize mouse IgG. The decalcified orbits were incubated with a combinational medium [10 wt% of Triton X-100 (#3051.1, Roth, Germany), 5 wt% of Tween-20 (#9127.2, Roth, Germany), 0.3 wt% of Albumin V (#0052.1, Roth, Germany), 50 mM of Glycine (#3908.3, Roth, Germany), and 0.05% of NaN3 in 0.2 M PBS] and shaken for 48 h at room temperature, in combination to delipide and block unspecific bindings. Then, the samples were washed in PBS of shaking for 24 h and stained with Cy3-conjugated Goat anti-mouse IgG (#115-165-062, Jackson ImmunoResearch Lab. Inc.) at a concentration of 15 μg/ml for 5 days at room temperature. The antibody was prepared in the diluted combinational medium with 0.1 M PBS (1:1). After staining, these samples were immersed and washed in 30% sucrose in PBS and subjected to embedding, and cryosectioning. As a negative control, no antibodies were applied. For the sections in which fluorochrome-conjugated antibodies were applied or those as the corresponding negative control, the mounting medium containing DAPI (#H-1200, Vector Labs) was used for coverslipping. As a control of a traditional way (e.g., WSA, without tape aided sectioning, staining, and aqueous mounting) of immunostaining, the sections were prepared after fixation, decalcification, embedding, and sectioning. After post-fixation with acetone for 10 min and unspecific blocking with a blocking medium [1% (w/v) of Albumin V (#0052.1, Roth, Germany), 25 mM of Glycine (#3908.3, Roth, Germany), and 2% (v/v) of goat serum (DAKO, Hamburg, Germany) in 0.01 M PBS] for 30 min, Cy3-conjugated Goat anti-mouse IgG was applied on the sections and incubated at 4°C overnight. The antibody was prepared in 0.01 M PBS at a concentration of 3 μg/ml.

The coronary sections used were collected at the level of Bregma, +3.90 (refer to Fig 3), and the regions indicated with (C) (in the image B of Fig 3) were viewed for comparison.

Fig 3

Images of immunostaining for IgG in orbits of native mice.

The sections of (A) are collected at the position of Bregma, +3.90 mm. B. The right orbital image magnified in the local of (A) displays a region (inset) of (C), in which the fluorescent images in the right panels (C) were taken. Bars indicate 50 μm.

Data analysis

Data are presented as means ± SEM. SPSS (IBM SPSS Statistics for Windows, Version 11.0., IBM Corp., USA) was employed for multiple comparisons of means. A p < 0.05 was considered statistically significant.

3. Results

Comparative evaluation of 8 methods

Tissue block staining and domestic adhesive tapes (e.g., BTA) produced qualified integral serial cryo-sections of whole orbits or eyeballs in adult mice, and the fine structures were well preserved (Fig 1). This method proved to be the best in comparison with seven other related existing histological traditional or non-traditional processing methods, according to the staining slice quality (Fig 4, for more detail, refer to S10 Table in Supporting Information in S1 File). Representative images processed by 8 techniques are shown in Fig 5. All the slice quality scores were compared using one-way ANOVA and Turkey post-test for multiple comparisons of SPSS, resulting in significant differences. A hundred percent success rate in sectioning, collection, and mounting with this method of BTA, is reflected in Table 2.

Fig 4

Slice quality comparison among different protocols.

The average slice quality scores of the 3 best staining sections at the position of Bregma +3.90 mm (the best quality section among 10 in one head of animal) in the 8 protocols (X-axis) are shown in Y-axis.

Fig 5

List of representative images of 8 protocols.

The representative best quality orbital and eyeball staining images for each protocol are shown at three objective magnification levels—macro (gross), X2.5, and X10. Images in Panel X10 and Panel X2.5 are the local magnifications in those of Panel X2.5 and Panel Macro respectively.

Table 2

Qualified staining section success rate.

BTA (%)	BTO(%)	BWA(%)	BWO(%)	TSA(%)	TSO(%)	WSA(%)	WSO(%)
100.0 (30)	93.3 (28)	80.0 (24)	76.7 (23)	83.3 (25)	80.0 (24)	73.3(22)	66.7(20)

Figures in the parentheses indicate the values of the qualified staining sections in 30 collected.

Reproducibility of BTA

A reliable method should be reproducible and not be affected by day-to-day affected variation. The reproducibility tested on qualified section success rates and slice quality scores with BTA (see the protocol in the Supporting Information in S1 File) were carried out in four different periods of time. To test the repeatability of the procedure of BTA, 8 heads of male DBA1/J mice (Janvier Labs, France), 33 weeks in age, were objected to the same processing as described above. The evaluations of slice quality scores and qualified section success rates (ZL) were completed in four separate experiments in different periods of time, with two heads for each. The results, as shown in Table 3, were in high similarity among four runs of measurements, which indicates a high reproducibility.

Table 3

Repeatability tests on slice quality scores and qualified section success rates with BTA in 4 separate experiments.

	First ran	Second ran	Third ran	Fourth ran
Success rate (%)	90 (18)	100 (20)	100 (20)	90 (18)
Quality scores (means±SEM)	10±2.02	12±2.02	11±2.02	10±2.02

The high qualified sections and success yield rates are obtained from these 4 different runs. 20 sections are collected each round. Qualified sections are counted as indicated in parentheses.

Application of BTA in immunostaining of orbits

In the eye research field, immunostaining of mouse orbits for cell-specific and molecular markers is critical. In order to extend the method’s potential application to immunostaining, we tried tissue block immunostaining in a simple and familiar way for a start. We selected the target of mouse IgG, which is often used in our laboratory to assess the severity of edema [44], as an example. On the other hand, there are abundant vessels including venous plexus or sinus and arteriolae around an eyeball in the normal state of native mice. IgG is trapped in the blocks of blood coagulation, which often occurs inside the vessels of the dead animals. Immunostaining with BTA for IgG was performed and the results demonstrated the target is present in the vessels (see Fig 3). As a negative control, there were no signals found at the corresponding position. In comparison with the immunostaining in the traditional way (e.g., WSA), a stronger signal was found in the image by BTA (see Fig 3). The preliminary outcomes displayed a stronger signal staining with BTA, which is at least comparable to the traditional immunostaining with WAS. Overall, we can conclude that immunostaining with BTA for molecular targets is feasible.

It should be stated that we cut the orbital tissues at a thickness of 7μm since it was difficult to obtain a complete 5μm-thick highly qualified section without aid of adhesive tapes during the preparation of the control (WSA). The higher resolution of the image (e.g., objective lens of > 20x) was limited due to the section thickness (7 μm).

Application of the method (BTA) in other technically sectioning-challenged tissues

Application of BTA in joints, brains, and so on,—the challenging integral sectioning tissues, also generated high-quality histological staining sections (refer to S6-S9 Figs in Supporting Information in S1 File).

4. Discussions

Generating high-quality histological sections of animal tissues can be technically challenging when using traditional staining protocols that preserve tissue integrity of morphology. These challenges are especially obvious when cutting thin sections (less than 10 μm in thickness) of whole orbits or eyeballs from adult animals. Even with every effort in sectioning, the sections displayed curling, distortion, and missing, as seen in traditional staining, e.g., WSO of Figs 4 & 5, and remained so although the lens had been removed prior to sectioning in some cases.

These phenomena are due to markedly various shrinkage of different tissues under different temperatures [34]. As soon as contact between the warm slide and the frozen section during cryo-sectioning is made, the section will immediately melt, which will then convert the ice crystals back into the water. The flow of the melted solutions can distort and displace fine structures because of different shrinkage. At this point, the melted section is dried and surface tension forces continually distort, displace, and collapse tissue structure. For example, the retina detached from the choroid or a huge gap between retina and choroid was often observed after sectioning. Even by chance, an intact section of orbital or eyeball is secured, but it becomes incomplete or displaces after conventional staining. The same phenomenon was observed during section flattening and collection when the paraffin sections float on the warm water. Thus, histological preparation of only parts of the orbital or eyeball has become a way to overcome at least some of the problems.

To preserve the integral and fine tissue structures, an alternative is to use adhesive tape or film. The adhesive on the sticky tape behaves both like a solid and a liquid. Like a liquid, it flows finding its way into any tiny gaps in the surface it is being stuck to when a little pressure is applied on it. It turns back into a solid when the pressure is withdrawn, allowing it to lock into those gaps and to hold itself in place [45]. Thus, the adhesive tape would help to stabilize, support, and capture sections.

Therefore, we used an adhesive tape-aided sectioning technique and traditional staining protocols in the orbital or eyeball tissue. In this procedure, adhesive tape was attached to cutting tissue surfaces, and when the block was sectioned and detached, the cut section remains adhered to the tape without curling or other deformation. As seen in TSO of Figs 4 & 5, however, although the staining section quality was much better than those of WSO, undesirable artifacts were still found. These artifacts included displacement and wrinkles of collagen in the interior wall of the orbital.

Contacts of the section-tape with these oil-soluble reagents such as ethanol or xylol during staining, dehydrating, clearing, and mounting was supposed to contribute to weakened bonding forces between section and adhesive tape [46], which will lead to distorting the intact sections as seen in TSO in Figs 4 & 5. Staining secondary to tape-aided sectioning as in TSO means that the tape—sections are contacted with oil-soluble solutions.

To circumvent the contact between the sections and adhesion diminished oil-soluble reagents, we developed a method of using a tissue block staining before the adhesive tape-aided sectioning, staining and dehydrating and clearing and oil-soluble mounting as the traditional staining procedures, allowing for intact cryo-staining-sectioning and high-quality staining sections as seen in Figs 1, 4 and 5 and S10 Table in S1 File.

According to the staining section quality score, the protocol of BTA is best in comparison with 7 other related existing protocols (refer to Fig 4). These 7 methods included either traditional methods of sectioning (with or without tape aid)–staining—mounting or nontraditional ones of block tissue staining—sectioning (without tape aid)—mounting or block tissue stained—tape-aided sectioning but mounting with oil-soluble medium (for more detail, see Figs 2 & 5).

Besides, with the protocol of BTA, the number of sections lost was greatly reduced after final coverslipping. 30 serial sections collected after sectioning yielded 30 intact and high-quality staining sections (refer to Table 2). The staining sections displayed high quality, free of folds or tears, and the faithful preservation of the fine structures (see Fig 1). The structure orientation was automatically achieved. This feature becomes important in applications requiring exact alignment of consecutive sections, such as comparisons among the different treatment groups and researches on the specific local site modification of retina in visual neuroscience It is most applicable to large, high-quality, and thin (<10 μm) serial sections, such as for 3-D digital reconstruction.

The repeat measurements on section quality scores and qualified section success rates for BTA protocol, as shown in Table 3, were in high similarity among the four runs. The high reproducible data confirmed that the technique is reliable over time.

Application with this protocol in the challenging integral sectioning tissues, such as joints (S6 Fig in S1 File), brain (S7 Fig in S1 File), heart or kidney (S8 Fig in S1 File), and lung or spleen (S9 Fig in S1 File) also generated high-quality histological sections. The cell structure with the protocol of BTA was well preserved without significant distortion.

The method’s potential application in immunostaining to detect molecular markers was also successfully implemented in our preliminary experiments. Its successful application in immunostaining will benefit somewhat in eye research.

In addition, HE is the most commonly used stain for light microscopy in histopathology laboratories due to its comparative simplicity and ability to demonstrate a wide range of both normal and abnormal cell and tissue components [47]. Whole intact sections and good quality images at relatively high magnification are essential for digital pathology [48]. Therefore, this method is also very suitable for analyzing whole slide images in digital pathology and thus able to provide global information for quantitative and qualitative image analysis. Digital pathology is a rapidly growing field, offering such advantages as remote diagnostics and the application of image analysis to improve the efficiency of the decision process [48-50].

Turnaround speed, section thickness, and tissue block size

The protocol of BTA originally designed to prepare orbital or eyeball staining sections, is also excellent for the preparation of other tissue staining sections. Using the protocol of BTA, it normally takes 3 or 4 days (exclusive of 1 day for tissue fixation and 21 days for decalcification) to acquire qualified staining sections from a tissue block. Sections as thin as 5 μm or above can be reliably prepared for the present setup. The largest specimens cut in the present study have been for coronal sections of an adult mouse brain, which is encompassed by a 15 x 25 x10 mm3 block. The block size can be increased considerably more than this.

Limitations

It should be stated that this technique (BTA) was found unsuitable for the liver, in which hematoxylin penetrated two millimeters in-depth (5 mm in total thickness) within 18 hrs of staining. Prolongation of incubation in hematoxylin solution or thinner thickness of the tissue block will be necessary for the liver. In addition, a single staining method (e.g., HE) with this technique is sometimes a drawback if a tissue requires special staining besides HE. But in general, HE is sufficient for the examination of eye histology [33]. Like other aqueous mounting ways, the staining sections with this method should be examined and photographed in 3 weeks, otherwise, they will be dry over 4 weeks. Should this occur, the staining sections can be retrieved by impregnation of one or two drops of aqueous mounting media on the border of the coverslip. Uneven HE staining, which sometimes occurs, may prevent the use of these sections for morphometric studies. But if the appropriately staining and decoloration processes are strictly controlled, the phenomenon can be avoided.

In summary, we have developed a robust cryo-histological method that allows imaging of the whole orbital or eyeball staining sections while maintaining excellent cellular and subcellular morphology. Its applications are not limited to orbit or eyeball, brain, and joints, and to HE staining.

Supporting information.

Complementary information of step-by-step protocol of BTA (Block staining, tape aided sectioning, and aqueously mounting), applications of BTA in the tissues rather than orbits, and original observational data.

(DOCX)

Click here for additional data file.

18 May 2021

PONE-D-21-05943

Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs

PLOS ONE

Dear Dr. Li,

Thank you for submitting your manuscript to PLOS ONE. I would like to apologize for how long it has taken to provide you with reviews.  After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The major concerns of the reviewers concerned your statistical analysis and the quality of the images:  You should use ANOVA followed by a post hoc test for multiple comparisons, rather than t tests.  As far as the images are concerned, the reviewers may have been interpreting the pdf versions, rather than the high definition tiff images that can be downloaded from the website.  However, reviewer 2 points out that fluorescence microscopy and immunofluorescence (IF) staining of rodent orbits with cell-specific and molecular markers are critical. The authors should assess the morphological outcomes of immunofluorescence staining with this method,

Please submit your revised manuscript by Jul 02 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Alfred S Lewin, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. To comply with PLOS ONE submissions requirements, please provide methods of sacrifice in the Methods section of your manuscript.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

4. Thank you for stating the following in the Financial Disclosure section:

[The author(s) received no specific funding for this work.].

We note that one or more of the authors are employed by a commercial company: Advancecor GmbH

Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Li and colleagues present a study comparing eight cryo-histological protocols for both quality and repeatability. The article is well written, the methods are clearly described, and the data will be beneficial for the scientific community. Though the paper is directed towards identifying the BTA method as both the most reproducible and highest quality, the authors present images of tissue from all eight methods in the supplemental data. Perhaps this figure could be moved to the main part of the manuscript, as it is central to the hypothesis tested in this study. These qualitative impressions would pair nicely with the quantitative data in Figure 3. My only concern of any significance is that it would appear that a student-t test was done in which the score from each technique was compared to the BTA technique. If the experimental design was to prepare tissue with eight different methods, evaluate the sections and identify which method produced the best result, a student-t test would not be the correct statistical tool. Perhaps a one-way ANOVA with a post-hoc Tukey-Kramer multiple comparisons test would be more appropriate?

Specific Comments:

Figure 1, 4, 5: The image quality in Figure 3 was acceptable. The image quality in Figure 1 is so poor I cannot evaluate the tissue. The image quality in 4, 5, and 6 is better than Figure 1, but still not great. Please increase the resolution. The images in the Supplemental Materials were of high quality.

Figure 2: This is extremely helpful and a wonderful visual aid towards providing the reader with a visual explanation of the experimental design.

Figure 3A: BTA – would the scale bar appear more clear in black? Perhaps this is why it not aligned with the other scale bars.

Figure 3B: Please label the y-axis. If 12 was the largest value (perfect score), why does the graph go to 14?

Supplemental Table 3: Are the quality scores presented an average? If so, please provide the SEM.

The data points behind the means were not provided in this study, which I believe is required (Figure 3/Table2/Table S3).

Additional comments:

Line 68 there is an extra space between “easy” and “collection”

Reviewer #2: The manuscript entitled “Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs” describes a new frozen sectioning method adapted to keep the morphology of rodent orbit morphology intact. The authors systematically evaluate the outcomes of routine H&E-stained sections with this new method compared with those obtained with other histology methods.

Major concerns relate to insufficient validation of the proposed approach.

1. In the eye research field, immunohistochemical staining techniques with cell-specific and molecular markers are critical. The authors should assess the morphological outcomes of immunoperoxidase stained with this new method, and compare TSA (Tape aided sectioning, staining, and aqueous mounting medium) and WSA (without tape-aided sectioning, staining, and aqueous mounting medium).

2. In the eye research field, fluorescence microscopy and immunofluorescence (IF) staining of rodent orbits with cell-specific and molecular markers are critical. The authors should assess the morphological outcomes of immunofluorescence staining with this new method, and compare TSA (Tape aided sectioning, staining, and aqueous mounting medium) and WSA (without tape aided sectioning, staining, and aqueous mounting medium). If IF is affected, the results should be disclosed with alternate techniques.

3. The authors should determine whether the use of 15% EDTA for 21 days for decalcification of the whole head specimens affects the immunostains. The use optimizes the concentration and duration of immersion with EDTA for immunostaining.

4. Lines 8 and Line 42, "OCT (optimal compound of tissue)" should be "OCT (optimal cutting temperature) compound".

5. Line 213, To compare the quality of section staining, only the best 1 out of the 10 serial sections at the position Bregma+3.90mm was selected for evaluation. They should perform the analysis of the sections at 5 different positions (Bregma +2.60, +3.25, +3.90, +4.55-, and +5.20-mm) as the serial quality of the sectioning is critical for 3D reconstruction.

6. The optical resolution of all the figures is suboptimal photos with better resolution should be provided.

7. The authors should disclose the rate of section loss and the rate of tissue folding. Table 2 showed the success rate for each of the 8 groups. If more than half of the stained section area is damaged or covered by folded tissue, it should be counted as section loss.

8. The authors should add a video as supplementary information.

9. Line 38. The authors should change “…serial whole integrated sections

of orbits or eyeballs to determine further precise site-specific morphological changes that respond to treatments or genetic modifications.” to “…serial whole sections

of orbits or eyeballs to determine morphological changes that respond to treatments or genetic modifications.”

10. Line 43. The tissue thickness should be disclosed.

11. Line 113. The authors should disclose the 3 dimensions of the tissue blocks.

12. Line 117: High concentration of saturated picric acid represents a biosafety risk. An alternate solution should be provided.

13. Line 140, The dimensions of the slide of glass and supplier should be disclosed.

14. Line 170 The authors should write the full name of BTA here.

15. Line 213. The authors state that “One best quality section of the right-side orbital tissues among the ten for each head was selected for section quality comparisons among the various protocols according to staining section quality.” The authors should justify, and explain whether the selection process was performed in a masked fashion.

16. They should indicate whether the person who performed the sectioning was different or not.

17. Line 228: They should indicate the person(s) who assessed the sections with initials.

18. The authors should disclose freezing artifacts such as vacuolization at high power in soft tissue such as retina that can be noticed at high power (X 40 objective). They should also disclose the distribution of freezing artifacts. The authors should show high power images of the deeper structures such as the retina (X 40 objective).

19. The results section should be separated from the discussion section.

20. The results with tissue types other than eye and orbit tissues should be excluded from this manuscript, as the results are preliminary and the quality of the sections has not been validated.

21. Line 367: The authors should disclose the total duration of processing from sacrifice to sectioning.

22. In the limitations section the authors should include uneven HE staining that may prevent the use of these sections for morphometric studies.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Jun 2021

Reviewer #1

COMMENT 1:

Li and colleagues present a study comparing eight cryo-histological protocols for both quality and repeatability. The article is well written, the methods are clearly described, and the data will be beneficial for the scientific community.

RESPONSE:

Thanks for the reviewer’s inspiring comments.

COMMENT 2:

Though the paper is directed towards identifying the BTA method as both the most reproducible and highest quality, the authors present images of tissue from all eight methods in the supplemental data. Perhaps this figure could be moved to the main part of the manuscript, as it is central to the hypothesis tested in this study. These qualitative impressions would pair nicely with the quantitative data in Figure 3.

RESPONSE:

It is a good suggestion. We have moved the figure to the main part of the manuscript. Please refer to Lines 296 – 303 in blue.

COMMENT 3:

My only concern of any significance is that it would appear that a student-t test was done in which the score from each technique was compared to the BTA technique. If the experimental design was to prepare tissue with eight different methods, evaluate the sections and identify which method produced the best result, a student-t test would not be the correct statistical tool. Perhaps a one-way ANOVA with a post-hoc Tukey-Kramer multiple comparisons test would be more appropriate?

RESPONSE:

We are agreed on reviewer’s points. A one-way ANOVA with a post-hoc Tukey multiple comparisons (unfortunately, there is no such item - Tukey-Kramer found in our SPSS software – V11. Sorry for that) have been made, which replaced the Student T test. The changes have been present in the current manuscript. Please see Lines 277 -280, 291 -294,296 - 298 in blue.

COMMENT 4:

Figure 1, 4, 5: The image quality in Figure 3 was acceptable. The image quality in Figure 1 is so poor I cannot evaluate the tissue. The image quality in 4, 5, and 6 is better than Figure 1, but still not great. Please increase the resolution. The images in the Supplemental Materials were of high quality.

RESPONSE:

The reviewer is correct. The images with a higher resolution – TIFF images are available in the new manuscript. Please refer to TIFF images – Figs 1,3,5 of the revision.

COMMENT 5:

Figure 2: This is extremely helpful and a wonderful visual aid towards providing the reader with a visual explanation of the experimental design.

RESPONSE:

Thanks!

COMMENT 6:

Figure 3A: BTA – would the scale bar appear more clear in black? Perhaps this is why it not aligned with the other scale bars.

RESPONSE:

The viewer is right. We have changed that scale bar with a black one. But we left Fig 3A out in revised manuscript to avoid needless duplication since all the eight resultant representative images have been moved into the main body of the manuscript according to the reviewer’s nice suggestion (Comment 2).

COMMENT 7:

Figure 3B: Please label the y-axis. If 12 was the largest value (perfect score), why does the graph go to 14?

RESPONSE:

Yes, we have labeled Y- axis and deleted the value of 14 in that Figure. Please note that the figure 3B in earlier version has changed into Figure 4 in the revision due to insertion of new images. Please refer to Lines 296 – 298 in blue.

COMMENT 8:

Supplemental Table 3: Are the quality scores presented an average? If so, please provide the SEM.

RESPONSE:

Yes, the quality scores present average values. We have added SEMs to each corresponding average value. Please see Lines 319 – 320 in blue.

COMMENT 9:

The data points behind the means were not provided in this study, which I believe is required (Figure 3/Table2/Table S3).

RESPONSE:

The reviewer is right. The complementary data have been added in the corresponding tables or figure of the revised manuscript. Please refer to Fig 4, Tab 2 and Tab 3 in revised manuscript and Lines 296 – 298, 305 -307, 319 – 320 for details in blue.

COMMENT 10:

Line 68 there is an extra space between “easy” and “collection”

RESPONSE:

Yes, correction has been made in Line 69 of the revised manuscript.

RESPONSES TO EDITORIAL AND REVIEWER COMMENTS

Reviewer #2

COMMENT 1:

The manuscript entitled “Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs” describes a new frozen sectioning method adapted to keep the morphology of rodent orbit morphology intact. The authors systematically evaluate the outcomes of routine H&E-stained sections with this new method compared with those obtained with other histology methods.

Major concerns relate to insufficient validation of the proposed approach.

1. In the eye research field, immunohistochemical staining techniques with cell-specific and molecular markers are critical. The authors should assess the morphological outcomes of immunoperoxidase stained with this new method, and compare TSA (Tape aided sectioning, staining, and aqueous mounting medium) and WSA (without tape-aided sectioning, staining, and aqueous mounting medium).

2. In the eye research field, fluorescence microscopy and immunofluorescence (IF) staining of rodent orbits with cell-specific and molecular markers are critical. The authors should assess the morphological outcomes of immunofluorescence staining with this new method, and compare TSA (Tape aided sectioning, staining, and aqueous mounting medium) and WSA (without tape aided sectioning, staining, and aqueous mounting medium). If IF is affected, the results should be disclosed with alternate techniques.

RESPONSE:

We are agreed on reviewer’s points. The integral sections of orbits are prerequisite for all the subsequent kinds of staining and morphological evaluation. The first step is to ensure an intact qualified section, which was originally designed for in the method, and the applications of the method are the next steps.

In order to extend its potential application in immune-staining, as reviewer’s kind suggestions, we made immunostaining in blocks of orbital tissue. That is a new issue, which involves the antibody permeability during 3D immunostaining (Susaki, 2020 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15906-5). Delipidation is necessary for the antibody penetration, but it may damage the cell membrane structures (concerning cell-specific markers). Therefore, we selected the target of mouse IgG, which is simple and often used in our laboratory to assess the severity of edema, (Li, 2020 Scientific Report | https://doi.org/10.1038/s41598-020-76950-1) as an example. The modified method from the present one described was adopted for the new application.

We cut the orbital tissue at a thickness of 7µm since it was difficult to cut a complete qualitified 5µm-thick-section without aid of adhesive tapes in the control (WSA). It is the section thickness (7µm) that an image of a higher resolution (objective lens > 20) was limited. But the images of a lower resolution, which is present in the revision, do not affect our conclusion that BTA application in orbital immunostaing is feasible. For more details, please refer to Lines 188 -189, 243 – 274, 322 -341 in red.

We have tried to make immunostaining with HRP-conjugated antibody, but the results were left out in the revised manuscript because of a little too weak signals against background. Here we express our deep regret.

COMMENT 2:

3. The authors should determine whether the use of 15% EDTA for 21 days for decalcification of the whole head specimens affects the immunostains. The use optimizes the concentration and duration of immersion with EDTA for immunostaining.

RESPONSE:

EDTA (15%) for 21 days or more or less for decalcification is often adopted before sectioning in osteology, odontology and the tissues with calcification. The treatment might affect the antigenicity of the targets for some or maybe not for the others. That is a big issue since there are huge number of different antibodies used for these diversities of the antigens. The different combines of EDTA and fixatives will make the situation more complicated. Our unit is rather small. The limited financial resource cannot afford the investigation at this moment. We would like to express an apology for this.

COMMENT 3:

4. Lines 8 and Line 42, "OCT (optimal compound of tissue)" should be "OCT (optimal cutting temperature) compound".

RESPONSE:

Thanks for the reviewer’s kind reminder. The corrections have been made in the manuscript. Please refer to Lines 8 – 9, 42 in red.

COMMENT 4:

5. Line 213, To compare the quality of section staining, only the best 1 out of the 10 serial sections at the position Bregma+3.90mm was selected for evaluation. They should perform the analysis of the sections at 5 different positions (Bregma +2.60, +3.25, +3.90, +4.55-, and +5.20-mm) as the serial quality of the sectioning is critical for 3D reconstruction.

RESPONSE:

The reviewer is right. To address the reviewer nicely suggestions, we reassessed the quality of sections at other positions (Bregma +2.60, +3.25, +4.55, and +5.20 mm). The observational outcomes were listed in Supporting Information. Please refer to S10 Table in Supporting Information.

COMMENT 5:

6. The optical resolution of all the figures is suboptimal photos with better resolution should be provided.

RESPONSE:

Yes. The images with a higher resolution – TIFF images are available in the new manuscript. Please refer to TIFF images – Figs 1,3,5 of the revision.

COMMENT 6:

7. The authors should disclose the rate of section loss and the rate of tissue folding. Table 2 showed the success rate for each of the 8 groups. If more than half of the stained section area is damaged or covered by folded tissue, it should be counted as section loss.

RESPONSE:

Yes. We have a new complementary table included in Supporting Information. Please see and also refer to the revised Table 2 (Lines 305 – 307) and S10 Table in Supporting Information.

COMMENT 7:

8. The authors should add a video as supplementary information.

RESPONSE:

We consent to the reviewer’s suggestions. A video will make readers understand the method in a simple and straight-forward way. Unfortunately, our poorly editing technique cannot satisfy the needs of a high resolution and clear images. Instead, we made the presentation, which showed more details with a rather high resolution, to complement the regret. Please see Step-by-Step protocol in Supporting Information.

COMMENT 8:

9. Line 38. The authors should change “…serial whole integrated sections

of orbits or eyeballs to determine further precise site-specific morphological changes that respond to treatments or genetic modifications.” to “…serial whole sections

of orbits or eyeballs to determine morphological changes that respond to treatments or genetic modifications.”

RESPONSE:

OK. The change has been made in the revised manuscript. Please see Line 39 in red.

COMMENT 9:

10. Line 43. The tissue thickness should be disclosed.

RESPONSE:

Yes. The tissue thickness (the reviewer might mean “ the section thickness”?) should be mentioned there. But we cannot find out the detail value in the author’s book. Sorry we can not offer the value. In general, the value is between 5 – 7 µm.

COMMENT 10:

11. Line 113. The authors should disclose the 3 dimensions of the tissue blocks.

RESPONSE:

Yes. We inserted the values of the 3D in the new manuscript. Please see Line 121 in red.

COMMENT 11:

12. Line 117: High concentration of saturated picric acid represents a biosafety risk. An alternate solution should be provided.

RESPONSE:

Yes. We acknowledge a risk using Boun’s fixative, - one saturated picric acid containing solution. But the risk will be kept at a minimum when all the operation related is performed in a hood. Actually, in our trial tests, we have optimized the fixatives. The outcome indicated that the best combination was one between block staining and Boun’s fixative, and suboptimal one is between buffer formaldehyde and block staining. Therefore, an alternate solution should be the fixative of buffer formaldehyde (4%, v/v). Please note that formaldehyde also bears a rather high biosafety risk. Please see Lines 119 – 120 in red.

COMMENT 12:

13. Line 140, The dimensions of the slide of glass and supplier should be disclosed.

RESPONSE:

Yes. We inserted the values of the 3D in the new manuscript. Please see Line 143 in red.

COMMENT 13:

14. Line 170 The authors should write the full name of BTA here.

RESPONSE:

Yes. We have added the full name of BTA there in the new manuscript. Please see Line 176 in red.

COMMENT 14:

15. Line 213. The authors state that “One best quality section of the right-side orbital tissues among the ten for each head was selected for section quality comparisons among the various protocols according to staining section quality.” The authors should justify, and explain whether the selection process was performed in a masked fashion.

RESPONSE:

Yes. The selection process was performed in a masked fashion. Please see Line 240.

COMMENT 15:

16. They should indicate whether the person who performed the sectioning was different or not.

RESPONSE:

Yes. It is the same person who performed the sectioning. But the person is blind to the treatments. What we did facilitates the comparisons in a unified set of criteria. Please see Lines 240,315 in red.

COMMENT 16:

17. Line 228: They should indicate the person(s) who assessed the sections with initials.

RESPONSE:

Yes. We have included the relative initials in the new manuscript. Please see Lines 240,315 in red.

COMMENT 17:

18. The authors should disclose freezing artifacts such as vacuolization at high power in soft tissue such as retina that can be noticed at high power (X 40 objective). They should also disclose the distribution of freezing artifacts. The authors should show high power images of the deeper structures such as the retina (X 40 objective).

RESPONSE:

We would like to express our regrets. We cannot distinguish the vacuoles due to embedding, freezing, staining, coverslipping and photographing artifacts, or real structures of its. A high resolution image of the retina with the depth (X 40 objective) has been inserted in revised manuscript. Please see D1 of Figure 1.

COMMENT 18:

19. The results section should be separated from the discussion section.

RESPONSE:

Yes, the separation between Results section and Discussion has been made in the new manuscript. Please see Lines 284,368.

COMMENT 19:

20. The results with tissue types other than eye and orbit tissues should be excluded from this manuscript, as the results are preliminary and the quality of the sections has not been validated.

RESPONSE:

Yes. We have excluded those parts from the main manuscript and moved to Supporting Information section.

COMMENT 20:

21. Line 367: The authors should disclose the total duration of processing from sacrifice to sectioning.

RESPONSE:

Yes. We have displayed the span from sacrifice to sectioning in the revised manuscript. Please see Lines 449 – 450 in red.

COMMENT 21:

22. In the limitations section the authors should include uneven HE staining that may prevent the use of these sections for morphometric studies.

RESPONSE:

Yes. We inserted the description in Limitations section of the new manuscript. Please see L466 -468 in red.

Submitted filename: response to reviewers.docx

Click here for additional data file.

15 Jul 2021

Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs

PONE-D-21-05943R1

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Alfred S Lewin, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciate the authors’ thought comments and responses to reviewer feedback. All significant concerns have been addressed. Of slight note, it appears there is a typo in the last sentence of the Abstract that I missed on the first review: Application of this protocol in joints, brains, and so on, - the challenging integral sectioning tissues, also generated high-quality histological staining sections.

Reviewer #2: The authors addressed all comments and queries.

Aminor suggestion:

Line 273 Bregman should be changed to Bregma.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

27 Jul 2021

PONE-D-21-05943R1

Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs

Dear Dr. Li:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Alfred S Lewin

Section Editor

PLOS ONE