Analyzing autophagosomes and mitophagosomes in the mouse brain using electron microscopy.

Electron microscopy (EM) is considered the gold standard for studying macroautophagy and mitophagy, essential cellular processes for brain health. Here, we present a protocol using EM to analyze autophagosomes and mitophagosomes in the mouse amygdala. We describe the preparation of brain sections, followed by staining and EM imaging. We then detail the steps to identify and analyze autophagosome-like and mitophagosome-like structures. This protocol can be easily adapted to analyze autophagosomes and mitophagosomes in other mouse brain regions. For complete details on the use and execution of this protocol, please refer to Duan et al. (2021).

Before you begin

All animal work needs to be approved by the Institutional Animal Care and Use Committee prior to beginning the experiment. The laboratory needs to be equipped with a chemical fume hood and have a specifically designated and authorized location for handling and storing radioactive materials.

Preparation one: fixative

Timing: 1 h

0.4 M phosphate buffer (PB)

Dissolve 56.0 g K2HPO4 and 10.6 g NaH2PO4 in Milli-Q or distilled water.

Bring the volume to 1000 mL with Milli-Q or distilled water.

Adjust pH to 7.4 with 1 M NaOH or 1 M HCl. Store at room temperature (20°C–22°C) indefinitely.

The following solutions should be prepared on the day of perfusion.

0.1 M PB with heparin

Dilute 0.4 M PB to 0.1 M with Milli-Q or distilled water.

Add heparin to a final concentration of 10 units/mL.

Fixative

Use 16% EM grade paraformaldehyde, 8% EM grade glutaraldehyde, and 0.4 M PB to prepare fixative containing 4% (v/v) paraformaldehyde, 2% (v/v) glutaraldehyde, and 0.1 M PB.

Preparation two: 0.1 M cacodylate buffer

Timing: 30 min

1 M cacodylate stock solution

Dissolve 42.8 g Na(CH3)2AsO2∗3H2O in Milli-Q or distilled water.

Bring the volume to 200 mL with Milli-Q or distilled water.

Adjust to pH 7.4 with 0.2 M HCl. Store at room temperature (20°C–22°C) indefinitely.

Preparation three: 0.3% lead citrate solution

Timing: 30 min

0.3% lead citrate solution

Add 80 mL distilled water over 0.3 g lead citrate.

Add 1 mL of 10 N NaOH and shake vigorously.

Adjust volume to 100 mL with distilled water.

Pass it through a 0.22 μm filter before use.

Always keep the cap closed to minimize CO2 contamination and store at 4°C. If the solution becomes white milky, it should be discarded.

Key resources table

Materials and equipment

Epon formula: Add all components in the order as they appear in the table. First, pour the EMBED812, DDSA, and NMA in order into a graduated 50 mL plastic centrifuge tube with a cap. Tighten the cap, then shake/rotate the tube gently by hand until the solution becomes homogeneous. Add the BDMA with a disposable pipette tip, then shake/rotate again to mix.

CRITICAL: Prepare fresh. Note that we also use some epon that has been stored at -20°C or -30°C in a sealed, 50 mL plastic centrifuge tube and warmed up to room temperature before use. Long-term cold storage may lead to partial polymerization, so it is only recommended if you wish, for the step using a mixture of epon and propylene oxide. Also, instead of a plastic centrifuge tube for storage of epon, one could use a sealed syringe to exclude air, i.e., to prevent condensation that may affect resin polymerization.

CRITICAL: Paraformaldehyde, glutaraldehyde, osmium tetroxide, uranyl acetate, BDMA, propylene oxide, EMBED 812, DDSA, and NMA are toxic. They are harmful if ingested, inhaled or in contact with skin. Uranyl acetate is radioactive. Propylene oxide is corrosive and a potential carcinogen. All these chemicals must be handled in a chemical fume hood by individuals wearing personal protection equipment (PPE) including at least lab coats and gloves (two layers of gloves for propylene oxide). All contaminated materials are to be disposed of as chemical waste or radioactive waste in compliance with regulations of hazardous waste.

Toluidine blue solution: Dissolve 500 mg toluidine blue and 1 g sodium tetraborate in 100 mL Milli-Q or distilled water.

CRITICAL: Wear lab coats and disposable gloves when preparing the toluidine blue solution. The toluidine blue solution can be stored at room temperature (20°C–22°C) indefinitely.

Step-by-step method details

Animal perfusion and brain sectioning

Timing: 2 days

The goal of this step is to fix the brain to preserve the structures of brains and organelles. High-quality perfusion is crucial for obtaining high-resolution electron micrographs. After perfusion, the brain is removed and cut with a vibratome into brain sections, which will be utilized in a later step to obtain ultrathin sections for EM. Care is taken during handling brain sections to avoid tissue damage.

Animal perfusion

Conduct perfusion in a chemical fume hood. Use the perfusion buffer (0.1 M PB containing 10 units/mL heparin) and the fixative (4% paraformaldehyde and 2% glutaraldehyde in 0.1 M PB) at warm room temperature (23°C–37°C).

Transcardially perfuse mice and extract the brains with a standard protocol. First, anesthetize mice with isoflurane and then inject them intraperitoneally with ketamine (110 mg/kg) and xylazine (7 mg/kg).

Wait until adequate anesthesia is achieved as determined by loss of response to toe pinching. Place the mouse on a shallow tray.

Make a small incision in the diaphragm beneath the rib cage using a pair of iris scissors.

Cut the diaphragm along the entire length of the rib cage to expose the pleural cavity.

Take care to avoid damaging the liver and the heart.

Insert a blunt tip needle into the left ventricle of the heart.

Use a hemostat to clamp the heart to secure the needle and prevent leakage.

Cut open the right atrium with a pair of iris scissors.

Attach the needle base to a three-way valve connected to two 50-mL syringes, each containing 0.1 M PB with heparin or fixative.

Turn the valve to open the PB line and manually inject 25 mL 0.1 M PB with heparin within 1 min.

The liquid coming out of the atrium should be clear and the lungs and liver should have become very light after perfusion with PB.

Switch the perfusion solution to fixative and manually inject 25 mL fixative within ∼4 min.

Isolate the brain.

Remove the head with a pair of scissors.

Expose the skull by making an incision along the midline from the neck to the nose. Remove the vertebrae and muscles that remain attached to the skull.

Use the tip of a pair of iris scissors to carefully cut open the skull. Lift up the blade when cutting to prevent damaging the brain. Use a rongeur to peel the skull away from the dorsal surface of the brain.

Use a spatula to sever the olfactory bulbs and nerves connected to the ventral surface of the brain.

Place the isolated brain in fresh fixative overnight (12–18 h) at 4°C.

Brain sectioning

Prepare a 5% agarose block. Add 5 g agarose powder and 0.85 g NaCl to 100 mL Milli-Q or distilled water. Heat the solution in a microwave oven until boiling. Pour the agarose solution into several 10-cm tissue culture plates and let it cool down.

Cut the agarose block into 3 × 1 × 1.5 cm (L × W × H) cubes.

After the overnight fixation, wash brains with PBS (3×20 min). Gently agitate brains on a rotating platform during incubation with PBS. Alternatively, PB may be used; PBS is used because it works as well for this step and is readily available in the laboratory. Note that during Step 2, keep the brain wet with PBS or PB at all times to avoid any drying artifacts.

Glue the agarose cube to the sample holder of the vibratome with Krazy glue (Elmer’s Products, Westerville, OH).

Make a clean, vertical cut along the rostral border of the cerebellum to remove the cerebellum, using a razor blade.

Place the brain on a piece of filter paper to dry the bottom surface of the brain; this surface should be only slightly moist to ensure that the glue sticks properly.

Glue the brain without the cerebellum to the sample holder of the vibratome against the agarose block using Krazy glue. Orient the brain with the dorsoventral axis in parallel with the holder plate and the rostral tip of the brain facing up.

Fill the cutting chamber of the vibratome with PBS. Cut 150-μm thick coronal sections. Transfer brain sections into 6-well plates with a fine brush.

Wash the brain sections with 0.1 M cacodylate buffer diluted from the stock solution (3×5 min), then proceed to Step 3 directly if time permits. Otherwise, keep brain sections at 4°C overnight (12–18 h) and conduct Step 3 on the next day.

Osmium tetroxide fixation, en bloc staining, dehydration, and resin infiltration

Timing: 1–1.5 days

Osmium tetroxide (OsO4) is a secondary fixative for electron microscopy; it also provides contrast staining. Uranyl acetate is used for contrast staining for electron microscopy; it also acts as a fixative. The brain sections are dehydrated with ethanol following secondary fixation and staining, then embedded in epon resins. Steps 3–4 need to be performed in a chemical fume hood.

Osmium tetroxide fixation

Prepare fresh 1% osmium tetroxide solution by adding 250 μL 4% osmium stock solution and 100 μL 1 M cacodylate stock solution to Milli-Q or distilled water. Bring the volume to 1 mL.

Remove cacodylate buffer from brain sections by using a disposable transfer pipette. Do not allow the brain sections to dry during any of the procedures in Step 3.

Add 1 mL or at least enough freshly made 1% osmium tetroxide solution to cover the sections, using a fresh pipette.

Incubate brain sections with the osmium tetroxide solution at room temperature (20°C–22°C) for 30 min in the dark.

Excessive light and heat may alter osmium tetroxide and reduce its effectiveness (Hayat, 2000; Woods and Stirling, 2008). Thus, we fix the tissue in the dark for a short time at room temperature; we prefer room temperature since osmium tetroxide penetrates the tissue slowly, and cooling might slow penetration even more. Alternatively, one can fix for longer in both dark and cold. However, it is not clear if light versus dark can make a significant difference considering these relatively short incubation times.

Wash brain sections 3×10 min with 0.1 M cacodylate buffer.

Dehydration and resin infiltration

Prepare 1% uranyl acetate solution by adding 0.01 g uranyl acetate to 1 mL of 50% ethanol. Protect the 1% uranyl acetate solution from light.

Dehydrate the brain sections with 50% ethanol (3×5 min).

Replace 50% ethanol with the 1% uranyl acetate in ethanol solution. Keep brain sections in the 1% uranyl acetate solution for 15 min in the dark.

Continue to dehydrate brain sections with 75% ethanol (2×5 min), 95% ethanol (10 min), and 100% ethanol (3×10 min).

Carefully transfer brain sections to 20 mL glass scintillation vials containing propylene oxide. After transfer, change propylene oxide once.

Replace propylene oxide with a 1:1 mixture of epon and propylene oxide. Incubate brain sections with the mixture for 1 h.

Replace the epon and propylene oxide mixture with epon. Keep brain sections in epon at room temperature (20°C–22°C) overnight (12–18 h).

On the day after dehydration, replace the overnight epon with freshly-made epon. Keep brain sections in epon for several hours (we keep them for 24 h) at room temperature (20°C–22°C).

Embedding, isolation and mounting of the amygdala

Timing: 3–4 days

This step is to prepare tissue blocks that will be cut into ultrathin sections. The researcher identifies the brain regions of interest under a light microscope and uses a scalpel to isolate them. The isolated epon-embedded tissues are mounted on cylinders made of epon.

Tissue embedding and isolation

Place dehydrated, epon infiltrated tissues between two Aclar sheets separated by a thin layer of epon to make an Aclar sandwich. Avoid trapping bubbles in the sandwich and cure it in an oven set at 64°C for 48 h to complete the embedding procedure.

Additional epon can be used to make the epon cylinders. Mount flat bottom embedding capsules (polyethylene; size “00”; Electron Microscopy Sciences, catalog number 70021) on a corresponding polypropylene capsule holder (6 pack from EMS; catalog number 70022-06). Pour the epon into the capsules and heat in an oven as above.

Remove the Aclar sandwich from the oven and cool it down at room temperature.

Remove one of the Aclar sheets. Then, under a light microscope, outline the amygdala with a marker pen; this area can be identified by comparing its shape and features to those seen in coronal sections in any standard atlas. Use a scalpel to carve out the amygdala along the marked outlines.

Tissue mounting

Prior to mounting tissues, use a razor blade to slightly scratch one end of the epon cylinder in a grid pattern to assist in adhesion of the tissue section.

Glue the epon-embedded amygdala to the center of the grid with one drop of Krazy glue placed on the side still covered with the Aclar material.

Ensure the tissue glued to the cylinder is flat and avoid using excessive glue. This usually produces a stable specimen for utrasectioning; however, if the tissue comes loose from the Aclar sheet, then the Aclar sheet can be removed and the tissue mounted directly to the cylinder.

Dry the cylinder mounted with the tissue at room temperature overnight (12–18 h).

Use a specimen holder supplied with the Ultramicrotome to keep the epon cylinder mounted with brain tissue in place and free both hands for trimming.

Trim the edges of the tissue to make it a trapezoid. Don’t damage the tissue.

Wipe off any debris from the cylinder with wet paper towels. Since the debris may contain stained tissue with heavy metals, it is preferred to wipe it off instead of blowing it off into the air.

CRITICAL: Use wood handles derived from cotton swabs to transfer epon-embedded tissues onto Alcar sheets. After the tissue is flattened on the first Alcar sheet, place one end of the second Alcar sheet on the first sheet and slowly lay down the rest of the second sheet to avoid bubbles. Illustrations of the epon cylinder and Aclar sandwich are shown in Figure 1.

Figure 1

Pictures of epon-embedded brain sections and epon cylinders mounted with brain tissues

(A) 150-μm coronal sections of mouse brain are shown embedded in epon, sandwiched between two layers of Aclar film; in the bottom image, pieces of the amygdala region have been cut out of the sections (arrows). Note that the sections are black due to treatment with osmium tetroxide.

(B) The piece of brain with the amygdala is mounted on an epon cylinder and then trimmed to form a trapezoid shape. The cylinder is then placed in a metal chuck (upper right image) and inserted in the ultramicrotome (not shown) for further sectioning.

Cutting ultrathin brain sections

Timing: 8 h

Cut thick brain sections

Cut thick sections (1 μm or more) from the face of the mounted brain tissue block on an ultramicrotome until the entire tissue face is exposed. We use a Diatome Histo Diamond Knife (45° angle, 6 mm; catalog number 60-HIS; Diatome U.S., Hatfield, PA; affiliated with EMS).

Stain these sections with a toluidine blue solution (0.5% toluidine blue [500 mg] plus 1% sodium tetraborate [1 g in 100 mL water]); we stain them on a hot plate and continue until the solution is dried.

Examine these thick sections under a light microscope to determine if the proper area has been reached for electron microscope examination, i.e., comparing the area to the known features as shown in a standard atlas.

Cut ultrathin brain sections

Cut ultrathin brain sections (60 nm) from the face of the mounted brain tissue with an ultramicrotome. We use a Diatome Ultra Diamond Knife (Wet; 45° angle, 3.5 mm; catalog number 35-US).

Mount the ultrathin sections on formvar/carbon-coated single-slot copper grids (EMS, catalog number FCF2010-Cu-SB). Alternatively, nickel grids (EMS, catalog number FCF2010-Ni-SB) may also be used.

Store grids in a grid box (EMS, catalog number 71147-12) in a desiccator until use.

The ultrathin brain section is shown in Figure 2.

Figure 2

Ultrathin-section cutting

(A) In the ultramicrotome, sections about 1-μm thick are cut, mounted on glass slides, and stained with a toluidine blue solution to visualize the subregions of the amygdala (BLA, basolateral amygdala; CeA, central nucleus of the amygdala).

(B) Low magnification electron micrograph of an ultrathin section correlating with a portion of the thick section in A. On the electron microscope, we first go to a low magnification (50–100×; imaged in LOW MAG mode without an objective aperture) and mark off the boundaries of the brain region on the ultrathin section to be imaged. This is done by correlating the image on the Gatan camera screen (Digital Micrograph program) with an image of the last 1 μm section taken before the ultrathin sections. Arrows in A and B indicate the same two blood vessel profiles.

(C) In the ultramicrotome, when the proper region has been reached, the ultrathin sections (60 nm) are cut and mounted on formvar/carbon-coated single slot grids (the hole is 1 × 2 mm); note that this ultrathin section is larger than the slot and so overlaps partly on the metal grid on the upper left side.

Electron microscope imaging

Timing: 4–8 h

Staining

We mount the grids on a Hiraoka grid support plate. The type that we use has been discontinued (see Figure 1 in Petralia and Wang, 2021), but a similar, modified Hiraoka grid support plate is available separately (EMS, catalog number 71560-32) or in the Modified Hiraoka Staining Kit (EMS, catalog number 71560-00).

Stain with 0.3% lead citrate solution (0.3 g lead citrate in 0.1 N NaOH; pass it through a 0.22 μm filter before use) for 3 min and then wash 3× with distilled water. Dry using a pipette to remove most of the water and then use the edge of a piece of filter paper to remove the last traces of water. Store in grid box until use.

Electron microscope imaging

Place a grid with a section of the brain in a holder and insert it into the JEOL JEM-2100 transmission electron microscope (TEM).

Align and examine at 200 kV.

This moderately high voltage allows electrons to penetrate the tissue more readily to improve image quality; and the JEOL 2100 maintains high image contrast at 200 kV as long as there is optimal sample preparation and thickness, due to its pole-piece with an in-gap, high-contrast objective lens aperture. Our lab and others routinely use the JEOL 2100 at 200 kV for all types of studies. For some other TEMs, one might need to use a lower kV to ensure adequate contrast.

Go to low magnification (50–100×) and compare the image on the Gatan camera monitor screen (using Digital Micrograph program) with an image of the last 1 μm section taken before the ultrathin sections (see Figure 2). Then mark off the boundaries of the brain region on the ultrathin section to be imaged, using the JEOL specimen position display window.

Take images at high magnifications (2,500× to 25,000×).

Expected outcomes

Every attempt is made to acquire an unbiased sample for quantitative analysis. During ultramicrotomy, we must take the thin sections from a very restricted and relatively small area in order to identify the boundaries of the amygdala nuclei accurately, so we are not able to utilize a wide area of the tissue to acquire random samples. However, the thin sections on grids from the different mice are assigned a code number unknown to the person who will image them on the TEM, so that the EM images are taken blindly. The imager marks off the boundaries of the relatively small area of the particular amygdala nucleus and then selects random areas within those boundaries only at low magnifications, so that the subcellular structures of interest cannot be discerned. Then, the imager goes to high magnifications on each of these randomly selected areas and captures the high-magnification images used in the study.

Electron micrographs are obtained after image acquisition and saved in the DM3 or DM4 format. The DM3/DM4 files are converted to JPEG files for image analysis. Subcellular structures can be resolved from electron micrographs at nanometer-resolutions. Autophagosomes and mitophagosomes are identified manually from electron micrographs. Subcellular structures with electron-dense contents and bound by a double limiting membrane are identified as autophagosome-like structures. Of these structures, those containing discernible mitochondria, as indicated by the presence of double membranes and presumptive cristae, are identified as putative mitophagosome structures; we call them mitophagosome-like structures to indicate that the identification of the cristae is not always definitive. Examples of autophagosome-like and mitophagosome-like structures are shown in Figure 3.

Figure 3

Electron microscopy

(A) Each grid is mounted on a grid-holder rod (arrow) that is inserted in the JEOL TEM, and tissue is imaged initially at lower magnifications to identify the proper regions for subsequent high-magnification imaging.

(B) High magnification TEM images of the basolateral amygdala (BLA) and central nucleus of the amygdala (CeA) showing examples of autophagosome-like and mitophagosome-like structures (arrows).

Quantification and statistical analysis

The experimenter is blind to the identity of the electron micrographs. The numbers of autophagosomes and mitophagosomes in each micrograph are counted manually. The total area of each micrograph is measured with commonly used image analysis software such as ImageJ, Metamorph, and Imaris. We count all autophagosome-like and mitophagosome-like structures in each micrograph and analyze all micrographs including those without autophagosome-like or mitophagosome-like structures in which the numbers of such structures are recorded as "0". Micrographs at the same magnification are used for quantitative analysis. The density of autophagosome-like and mitophagosome-like structures is calculated by dividing their total numbers in each micrograph by the area of the micrograph and averaged over ∼200 micrographs. Appropriate statistical analysis is conducted if the study goal is to determine the effect of experimental treatment on autophagosomes and mitophagosomes. Specific statistical tests are chosen based on the experimental design, number of experimental conditions, number of variables, sample size, and the null hypothesis to be tested.

Limitations

Autophagosomes and mitophagosomes are transient structures that are sensitive to the extracellular and intracellular environments affected by the animal's experience such as housing conditions, anesthesia, and behavioral testing. The quality of electron microscopy is dependent on the procedures of preparing brain tissues including animal perfusion, tissue fixation, and tissue staining. These biological and technical factors may have significant effect on the sensitivity and quantifiability of the method described in this protocol. Careful consideration of control groups, minimization of stress to the animal, standardization of animal housing, handling and behavioral testing, and quality check for each step of tissue preparation are warranted to achieve high-quality results. The numbers of autophagosomes and mitophagosomes in each cell are relatively small. An adequate number of images and size of imaging fields are necessary to obtain reproducible results for both discovery and quantitative studies. We typically collect ∼200 micrographs (5.6 × 3.38 μm) from 3 animals for each group. Because this protocol relies on visual inspection to identify mitophagosome-like and autophagosome-like structures, the data analysis step is time-consuming and needs experienced researchers proficient at identifying these structures from electron micrographs. These factors limit the throughput and efficiency of the EM-based analysis of macroautophagy and mitophagy.

Troubleshooting

Problem 1

Ultrastructural examination shows a lack of microtubules (step 1).

Potential solution

When fixing the tissue, make sure that the fixative initially is at room temperature or warmer to avoid loss of microtubules that may occur with initial fixation in cold fixative.

Problem 2

Poor ultrastructure including membrane damage and swollen or shrunken organelles (steps 1 and 3).

Keep the perfusion wash-out time short (less than 1 min) prior to adding the fixative to avoid loss of ultrastructure quality. Proper osmolality and pH of the fixative are also important to avoid swelling or shrinkage of organelles. It is best to use only sealed aliquots of paraformaldehyde and glutaraldehyde and make up the fixative just before use. The same is true for the osmium tetroxide solution. It should be a light color before use. If it is dark, it is likely bad and should not be used. The tissue should look evenly dark when the osmium tetroxide step is completed; if not, one may need to agitate the solution during this step. Poor osmium fixation can affect both tissue ultrastructure and contrast (see next problem). Make sure that the tissue is never allowed to dry during any of the solution changes in the fixation, sectioning, and dehydration procedures.

Problem 3

Sections under TEM show poor contrast (steps 9 and 10).

This could be due either to problems in section preparation or TEM operation. You might want to make up a new lead citrate solution. When making it, we generally keep the container closed at all times of the mixing, although it is not clear if this is absolutely necessary. We sometimes add an additional step of staining with 1% uranyl acetate for 5 min (followed by washing and drying) prior to staining with lead citrate. In the TEM, one can try using a smaller objective aperture. We noted that using JEOL 2100 at 200 kV gives good contrast, but those using other TEMs may need to use lower kVs such as 80–100.

Problem 4

Sections under TEM are contaminated with particles of various sizes and shapes (step 9).

In order to avoid lead contamination artifacts on the section, when staining with lead citrate, be careful not to allow any partial drying while washing with water. Also, make sure that the diamond knife and water trough on the ultramicrotome and all instruments are clean.

Problem 5

Borders of the desired brain region are unclear in the TEM (step 10).

One may overcome difficulties with setting the exact areas of brain regions (such as nuclei within the amygdala) by looking for clear markers such as blood vessels and irregular features visible on the thick section that is used to orient/correlate with the low magnification image seen with the TEM camera.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Zheng Li (lizheng2@mail.nih.gov).

Materials availability

This study did not generate new materials.

Reagent	Final concentration	Amount
K2HPO4	0.322 M	56.0 g
NaH2PO4	0.088 M	10.6 g
H2O	n/a	Adjust to 1000 mL of total volume

Reagent	Final concentration	Amount
paraformaldehyde	4% (v/v)	25 mL
glutaraldehyde	2% (v/v)	25 mL
0.4 M phosphate buffer	0.1 M	25 mL
H2O	n/a	25 mL

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Ketamine	VetOne	NDC 13985-584-10
Xylazine	Akorn	NDC 59399-110-20
Paraformaldehyde (16% aqueous)	Electron Microscopy Sciences	Cat#15710
Glutaraldehyde (8% aqueous)	Electron Microscopy Sciences	Cat#16019
Heparin	Sigma-Aldrich	Cat#9041-08-1
Sodium cacodylate trihydrate	Sigma-Aldrich	Cat#C0250
4% osmium tetroxide stock solution	Electron Microscopy Sciences	Cat#19170
Uranyl acetate	Electron Microscopy Sciences	Cat#22400
Propylene oxide	Polysciences	Cat#00236
BDMA	Electron Microscopy Sciences	Cat#11400-25
EMBED812	Electron Microscopy Sciences	Cat#14900
DDSA	Electron Microscopy Sciences	Cat#13710
NMA	Electron Microscopy Sciences	Cat#19000
Lead citrate	Electron Microscopy Sciences	Cat#17800
Agarose	LONZA	Cat#50005
PBS	Invitrogen	Cat#AM9624
Toluidine blue	Sigma-Aldrich	Cat#92-31-9
Isoflurane	Baxter	NDC 10019-360-40
Experimental models: Organisms/strains
Mouse: C57BL/6, 6 weeks to 5 months old, male	Charles River	Strain code: 027
Software and algorithms
Fiji-ImageJ	(Schindelin et al., 2012)	https://imagej.net/software/fiji/
Other
Leica EM UC7 Ultramicrotome	Leica Microsystems	n/a
Leica VT1200S Vibratome	Leica Microsystems	n/a
JEOL JEM-2100 transmission electron microscope	JEOL	n/a
Gatan camera screen (Digital Micrograph program)	Digital Micrograph	n/a
Three-way valve	Fisher Scientific	Cat#NC0209042
50-mL syringe	Fisher Scientific	Cat#13-689-8
Diatome Histo Diamond Knife (45° angle, 6 mm)	Electron Microscopy Sciences	Cat#60-HIS
Diatome Ultra Diamond Knife (Wet; 45° angle, 3.5 mm)	Electron Microscopy Sciences	Cat#35-US
Formvar/carbon-coated single-slot copper grids	Electron Microscopy Sciences	Cat#FCF2010-Cu-SB
Formvar/carbon-coated single-slot nickel grids	Electron Microscopy Sciences	Cat#FCF2010-Ni-SB
Grid box	Electron Microscopy Sciences	Cat#71147-12
Modified Hiraoka Staining Kit (alternative staining products)	Electron Microscopy Sciences	Cat#71560-00
Modified Hiraoka grid support plate	Electron Microscopy Sciences	Cat#71560-32
Iris scissors	World Precision Instruments	Car#14219

Reagent	Final concentration	Amount
EMBED812	45.87% (v/v)	20 mL
DDSA	0.287 M	12.5 mL
NMA	0.229 M	10 mL
BDMA	0.025 M	1.1 mL
Total	n/a	43.6 mL

Reagent	Final concentration	Amount
Toluidine blue	0.5% (w/v)	500 mg
Sodium tetraborate	1% (w/v)	1 g
H2O	n/a	100 mL

Reagent	Final concentration	Amount
Agarose	5% (w/v)	5 g
NaCl	0.85% (w/v)	0.85 g
H2O	n/a	100 mL

Reagent	Final concentration	Amount
4% osmium stock solution	1% (v/v)	250 μL
1 M cacodylate	0.1 M	100 μL
H2O	n/a	650 μL
Total	n/a	1 mL