Double staining immunohistochemistry.

This article summarized immunohistochemistry methods generally used in research laboratories and clinic including direct immune staining, indirect immune staining, enzyme method, fluorescence method, APC method and PAP method.

Introduction

Immunohistochemistry (IHC) is a useful research tool and used to localize specific antigens in tissue sections with labeled antibodies based on antigen-antibody interactions. The immune reactive products can be visualized by a marker including fluorescent dye, enzyme in general; radioactive element or colloidal gold also can be used.

Not only used in research laboratories, IHC is also used in clinical diagnosis; it has been one of the routine diagnostic approaches in daily clinic activities. Prior to the IHC generation, histochemistry was used frequently in clinical diagnosis and laboratory research. However, the histochemistry has been gradually replaced by IHC based on its apparently advantage, the specific antigen-antibody reaction. Theoretically, IHC can identify any existing protein antigen in tissue and thus make it become a critical technique in laboratory research and clinical diagnostic method.

In this article, we aimed to summarize several useful IHC methods that have been used in our laboratories for years. Tissue sampling, preparation, direct staining, indirect staining, double staining etc are described in this article in detail.

Tissue Preparation

Fixation

A principle of IHC tissue fixation is to keep minimal tissue structure and retain most antigenicity. We must ensure to preserve the readable tissue architecture and cell morphology; otherwise, the localization of immune reactive products can not be recognized. However, if tissue is over fixed, the antigenicity will be diminished or even completely extinguished that result in false negative staining.

There are a broad array of fixative method can be selective from. Researchers choose appropriate fixatives based on the tissue types and the requirement of experiments. However, we must say there is no one universal fixative that is ideal for the staining for all antigens. In general, most antigens can be successfully demonstrated in acetone-fixed tissue or sections. But formalin-fixed tissue sections also popularly used in IHC because formalin-fixed and paraffin-embedded tissue can be stored for many years. It is very convenient using archived paraffin-embedded tissue to carry out IHC. In recent years the use of antigen retrieval techniques boosted the application of formalin-fixed tissue to be used in IHC.

Common fixatives used for IHC

100% acetone. Pre-cold in a fridge. Cryosections are prepared, dry at room temperature overnight. Fixed with cold acetone for 20 min.

4% paraformaldehyde in 0.1M phosphate buffer. Tissue can be cut into 3 × 3 × 6 mm in size; fixed for 12 h at room temperature. Dry cryosections can be fixed at room temperature for 1 h.

2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer. Fixation is the same as (2).

PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer. Fixation is the same as (2).

4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistry). Tissue is cut into 2 × 2 × 4 mm, fixed for 2 h at 4°C.

Sectioning

Cryosections

Cryosections are the first choice of IHC. It is the best approach in terms to preserve the antigenicity for target antigens. The disadvantage of cryosection is that results in poor morphology, poor resolution in high power image. However, in general, cryosection derived image can still reflect tissue structure with required significance and has the capacity to satisfy publisher's requirement.

Making frozen blocks is the first step in cryosection (Fig. 1). Prepare some block molds that look like cylinder with about 1.5 cm in diameter, made with tinfoil paper, filled with Tissue Tek O.C.T. about 1-1.5 cm in depth. Sample tissue should be cut in appropriate size and placed in the OCT cylinder bottom in right orientation. The OCT cylinder then is immersed into liquid nitrogen for about 2-3 min until the liquid nitrogen not boiling. The blocks now can be stored at -80°C for future use or moved to cryostat to cut sections.

Fig. 1

Making OCT blocks. A-C, making a mold with tinfoil. D, fill the mold with OCT. E, insert tissue into the bottom of mold. F, an OCT block after frozen in liquid nitrogen.

Samples of muscle or mucosa can be cut in 4-6 micrometers; tissue of the brain and spinal cord needs to be cut in 10-40 micrometers. Crysections can be picked up by glass slides directly from cryostat; labeled properly and let dry at room temperature overnight, or, if urgent, the sections can be dried with a power fan within one hour.

After dry, sections can be wrapped with tinfoil and stored at -80°C for future use, or stored in a box with lid at 4°C temporally for up to one week, or, moved to staining stage.

Paraffin sections

The advantage of paraffin sections is that can preserve good tissue structure, but loose antigenicity from formalin fixation and the high temperature (60°C) during embedding. However, sometimes we do not have choice; for example, there is only paraffin embedded tissue available. Therefore, we have to deal with formalin-fixed tissue in IHC.

Cutting paraffin sections are relatively easier than cutting cryosections. One tip here is that you have to wait until the sections spread sufficiently in warm bath. Using a paper tissue wrap out the water bubble in warm bath frequently, otherwise the bubble may be trapped between sections and glass slides and becomes difficult to get rid off.

Paraffin sections need to be dewaxed with xyline and rehydrytion to water.

Whole Mount Preparation

Some tissue (less than 5 mm thick) such as mucosa can be examined by IHC as whole mounts. Whole mount preparations can show three-dimensional information about the location of antigens such as neurons and nervous fibers. Because of thick, it is difficult for antibody to penetrate into the tissue. Treated with Triton X-100 can increase the permeability and improve the staining results.

Antigen Retrieval

After fixation, the epitopes in tissue can be cross-linked and covered that makes it difficult for antibody-binding. By pretreatment with antigen retrieval reagent or procedures can significantly re-open the cross-linked epitopes so that antibodies can easily bind to target antigens. Several approaches have been proposed to retrieve antigens including heat-retrieving and enzyme-retrieving.

Heating retrieving

Steam heat retrieval: Place sections in a container containing citrate buffer (pH 6.0) and place the container in a steam device. Set the temperature to 100°C and let it boiling for 20 min.

Other devices like microwave oven, pressure cooker also can be used for this purpose.

Enzyme retrieving

Proteinase k, trypsin, chymotrypsin, pepsin, pronase and various other proteases can be used to retrieve antigens from formalin-fixed tissue. The concentration and treating time vary depending fixing time and concentrations. Users need test the condition in preliminary study.

Combination of heating retrieving and enzyme retrieving

This approach can be used as a second choice when either heating retrieving or enzyme retrieving does not work well. Again, the condition such as treating time and enzyme concentrations need to be tested in preliminary study.

Sodium borohydride method

Sodium borohydride (1% in phosphate buffer) treatment can be used to retrieve antigen from formalin fixed tissue. It is especially good at retrieve antigens from glutaradehyde fixed tissue. The treating time need to be tested prior to the experiment.

Note: Triton X-100 is not suitable using in antigen retrieving when known the antigens are localized on the surface of cells because Triton X-100 destroy membrane structure.

IHC Methods

Blocking

Several endogenous substances may interfere with IHC results, such as endogenous peroxidase, endogenous fluorescence, endogenous antibody binding capability (Fc receptors), endogenous biotin. Therefore, blocking the background staining is crucial in IHC to avoid acquiring false positive staining.

Blocking of endogenous enzymes

Endogenous enzymes such as AlkPhos, AcPhos and esterases can be destroyed by boiling, even a short time at 100 C.

Peroxidase is not blocked by boiling. It can be done by preincubating the slides in 3% H2O2 in 1% Sodium Azide PBS for 30 minutes plus several washes.

Peroxidase can be also blocked by 1.5% H2O2 in absolute methanol. Methanol incubation is also a fixation step that may affect your staining. According to the author's experience, method (2) is better than method (3). Fig. 2 shows IHC images from staining with (A) or without (B) blocking.

Fig. 2

Comparing IHC images with or without blocking. Intestinal segments were processed for IHC with or without blocking. Two representative images show the effect of blocking. The non-blocked image show the results of staining in which not only very dirty, but also no positive cells were stained.

Blocking of endogenous fluorescence

Blocking of endogenous fluorescence is very difficult. Pretreatment with UV for 20 min has some effect.

Blocking endogenous biotin

Endogenous biotin can be blocked with commercially available kits. The effective reagents in these kits are free avidin. Users also can by these components to block endogenous biotin. In addition, it can also be done with following procedures:

Wash 2-3 times in TBS-T (Tris 0.05M pH7.5; 0.01% Tween 20.

Add 1% ovalbumin on sections for 30 min.

Wash again with TBS-T.

Note: To block the endogenous biotin only when you use biotin-avidin system in your experiment. Otherwise, it is unnecessary.

Blocking of endogenous Fc receptor

Since Fc receptors are highly expressed on macrophages, B cells, T cells and other cell types that have the preferential avidity to bind antibodies using in IHC, blocking Fc receptor in tissue is necessary. In general, the Fc receptors in tissue can be blocked by incubation with 1% serum of the same species for 30 min.

Positive control

Positive control only used if the results are negative. It is used to test a protocol to make sure it works. You have to find a tissue that has known positive antigen of interest using as a positive control tissue. If the positive control tissue showed negative staining, the protocol or procedure must have problem and needs to be tested until a good positive staining is resulted.

Negative control

Omit the first antibody and add serum from host of the first antibody.

Add isotype IgG from host of the first antibody.

Adsorption of a primary antibody with the target antigen before use.

Direct Method

Direct method saves time, the detective marker (such as HRP, fluorescence) is labeled on the first antibody; but it is not as effective as indirect method because the signal is not amplified.

Indirect Method

Indirect method uses an unlabeled primary antibody (first antibody) against target antigen in tissue and a labeled secondary antibody. The second antibody reacts with the primary antibody (The secondary antibody is usually raised in animal by priming the animal with IgG from the host of primary antibody). Indirect method is much more sensitive than direct method because the detective signal is amplified by binding more than one molecules of the second antibody to the first antibody molecule.

The second antibody can be labeled with a fluorescent dye such as FITC, PE and Cy5 (indirect immunofluorescence method). The second antibody may be also labeled with an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase (indirect immunoenzyme method, see Figure 3 for images).

Fig. 3

Indirect staining results. Intestinal segments were processed for IHC to stain neural tissue with anti-PGP9.5 antibodies labeled with FITC or Cy5.

PAP Method (peroxidase anti-peroxidase method; Fig. 4)

This method is an extension of indirect immunoenzyme method. It adds a third antibody, anti-peroxidase antibody to the system. The third antibody binds to the peroxidase on the second antibody to form a firm complex. The advantage of PAP method is enhancing the dilution of the primary antibody, reducing background. The disadvantage is needed to pay more attention to the endogenous peroxidase; sometimes it raises the issue of non-specific staining.

PAP staining shows T cell and B cell in intestinal tissue. Colonic biopsy was processed for localization of B cells and T cells in the intestine by PAP method as described above.

Avidin-Biotin Complex Method

Avidin is a basic glycoprotein (MW 68 Kd) which has a high affinity for the small (MW 24Kd) water soluble vitamin biotin. Biotin can be conjugated to a variety of biological molecules, including antibodies, and many biotin molecules can be attached to a single molecule of protein. The biotinylated protein can thus bind to more than one molecule of avidin. The abbreviation “ABC” is stemmed from these three words: avidin-biotin complex. ABC method is actually a standard indirect method. We point it out here is because it has much better effect as compared with other indirect methods. The detective signal is highly amplified in the process. A disadvantage of using avidin is its non-specific binding to negatively charged structures such as the nucleus. Using streptavdin can reduce the non-specific binding.

Double IHC

Double IHC is actually the indirect IHC method. The difference is in double IHC, two sets of antibodies are used. The following is an example protocol for IHC.

Double IHC with confocal microscopy (protocol, see Figure 5 for image)

After fixing with acetone, sections are wash twice in TBST.

Block with 1-3% BSA or 1-3% goat serum for 30 min at RT.

Place the sections in a humidified box, add the first antibodies (antibody A is labeled by biotin; antibody B is not labeled), stay at RT for 1 h or 4°C overnight.

Wash with TBST (5 min, 3 times, 5 × 3).

Add fluorescence (such as FITC, PE or Cy5)-labeled streptavidin (for antibody A) and a second antibody (labeled with a fluorescence that has different weave length from the one labeled on streptavidin) against antibody B. Incubate at RT for 1h.

Wash (5 × 3).

Mount coverslip with anti-fading medium.

Observe with a confocal microscope.

Double staining colocalizes ChAT and 5-HTR2 on vagus neurons. Cultured vagus neurons were stained with anti-chloline acetyltransferase (ChAT) and anti-5-HTR2 antibodies with the procedures described above.