31.1.1 Materials

1. 
   

   Reagents: 0.01 M phosphate buffered saline (PBS, pH 7.4), bovine serum albumin, xylene, ethanol, 0.1% trypsin, microwave antigen retrieval solution (for heat method), primary antibody, secondary antibody, 3% hydrogen peroxide (H₂O₂), enzyme substrate (e.g., for peroxidase: avidin-biotin complex [ABC] kit, diaminobenzidine [DAB]), Mayer′s hematoxylin

   
   
2. 
   

   Materials: Paraffin-embedded tissue sections, microwave-resistant plastic staining container

   
   
3. 
   

   Equipment: Microwave oven, light microscope

31.1.2 Methods

1. 
   

   Removal of paraffin and rehydration

   
   
   
   
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     Dewax sections in xylene with two changes, 10 minutes each.

     
     
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     Rehydrate sections in graded ethanol (from 100 to 25%, 5 minutes each).

     
     
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     Rinse the slides in running tap water for 30 seconds.

     
     
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     Replace in PBS wash bath for further rehydration (30 minutes, room temperature).

   
   
2. 
   

   Antigen retrieval (either enzymatic digestion or heat-based)

   
   
   
   
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     Enzymatic digestion: Place slides in 0.1% trypsin in 0.1% CaCl₂ (pH 7.8) and incubate them at 37°C for 30 minutes. Rinse slides with 1 × PBS three times.

     
     
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     Heat-based: Wash the slides with deionized water and place them in a microwave-resistant plastic staining container with the slides fully covered by antigen retrieval solution. Operate the microwave oven at high power for 5 minutes; this process can be repeated two to three times. Cool down the slides at room temperature for at least 20 minutes.

   
   
3. 
   

   Inactivation of endogenous peroxidase

   
   
   
   
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     Place slides in 3% H₂O₂ in PBS at room temperature for 10 minutes.

     
     
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     Wash slides with 1 × PBS three times.

   
   
4. 
   

   Blocking

   
   
   
   
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     Cover slides with 3% bovine serum albumin and 5 to 10% normal serum (from the same species as the host of the secondary antibody) at room temperature for 1 hour or at 4°C overnight in a humid box, prior to primary antibody application.

   
   
5. 
   

   Primary antibody reaction

   
   
   
   
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     Drain the slides and carefully wipe each slide using a paper towel.

     
     
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     Dilute the primary antibody or negative control reagent in diluent to an optimal dilution, while diluent alone can serve as a negative control. A positive control slide (a tissue known to have the target antigen) should also be run.

     
     
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     Apply 100 µL primary antibody solution to cover the tissue sections and make sure the solution is evenly spread on the slides.

     
     
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     Incubate at 37°C for 60 minutes, at room temperature for 2 hours, or at 4°C overnight in humidified chamber. Longer incubation may be needed for low-density antigens.

     
     
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     Wash slides three times in 1 × PBS for 3 minutes each.

   
   
6. 
   

   Secondary antibody reaction

   
   
   
   
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     Drain the slides and carefully wipe each slide using a paper towel.

     
     
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     Dilute the biotinylated secondary antibody in diluent to an optimal dilution.

     
     
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     Apply 100 µL secondary antibody solution to cover the tissue sections and make sure the solution is evenly spread on the slides.

     
     
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     Incubate at room temperature for 1 hour in humidified chamber.

     
     
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     Wash slides three times in 1 × PBS for 3 minutes each.

   
   
7. 
   

   Color development

   
   
   
   
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     Prepare ABC reagent according to manufacturer′s instructions and apply to cover the sections. (Note: If secondary antibody is conjugated with peroxidase, ABC kit can be skipped; jump to DAB application.)

     
     
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     Incubate in a humidified chamber at room temperature for 30 minutes.

     
     
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     Wash slides three times in 1 × PBS for 3 minutes each.

     
     
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     Place slides in freshly prepared DAB solution and keep in dark for 2 minutes or longer to develop the color.

     
     
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     Wash the slides with deionized distilled water to stop the reaction.

   
   
8. 
   

   Counterstain

   
   
   
   
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     Using hematoxylin: Wash the slides with distilled water, place slides in hematoxylin at room temperature for 0.5 to 5 minutes (depending on strength of hematoxylin), rinse with distilled water, incubate slides in 0.5% NH₄OH (v/v) for 20 seconds, and rinse with tap water.

     
     
   • 
     

     Using methyl green: Wash the slides with distilled water, place slides in methyl green for 1.5 minutes at room temperature, rinse with distilled water, and dip slides 5 to 10 times in acetone with 0.05% acetic acid (v/v).

   
   
9. 
   

   Mounting

   
   
   
   
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     Dehydrate sections in graded ethanol (from 80 to 100%, 1 minute each).

     
     
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     Mount slides with hydrophobic mountant.

31.2.1 Choice of Antibodies: Polyclonal versus Monoclonal

Antibodies play critical roles in the success of IHC and therefore a careful selection of antibodies is essential. Antibodies information can be easily retrieved from online search engines and antibody manufacturers’ websites. The first and most common question for the choice of antibodies is the decision between polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs). There is, however, no absolute answer for this question, as this is determined by many factors. The most important one is its intended use and whether the antibody is readily available from commercial suppliers or researchers. In the meantime, the researchers should learn some facts about pAbs and mAbs (Table 31.1).

31.2.2 Polyclonal Antibodies

pAbs are produced by inoculation of animals and derived from different types of immune cells. The animals used are usually rabbits, mice, goats, chicken, horses, and guinea pigs, etc., where rabbits and mice are mostly used (pAbs raised from rabbits usually have higher affinity than from mice). The production cost of pAbs is relatively low and the time scale is short. pAbs have the features of recognizing multiple epitopes on an antigen. Therefore, pAbs show the following advantages and disadvantages.

Advantages: (1) more robust detection due to less sensitive to minor changes in the antigen, (2) a preferred option for denatured proteins, (3) may amplify signals from the antigens with low expression.

Disadvantages: (1) not useful for probing specific domain of antigen, (2) relatively high batch-to-batch variability, (3) may give high background signals due to certain extent of nonspecificity.

31.2.3 Monoclonal Antibodies

mAbs are produced by hybrid cells (hybridoma; i.e., mammalian cells fused with endlessly replicating tumor cells) and derived from a single cell line, whereas mouse and rabbit mAbs are more common. The production cost of mAbs is expensive and the time scale for hybridomas is long. mAbs have the feature of recognizing one epitope on an antigen. Therefore, mAbs show the following advantages and disadvantages.

Advantages: (1) high purity and good consistency when all batches are identical, (2) usually give rise to low background signals due to low cross-reaction with other proteins, (3) can generate reproducible results due to high homogeneity.

Disadvantages: (1) more sensitive to the minor changes in the antigen but this can be improved by using two or more monoclonal antibodies recognizing different epitopes on the same antigen, (2) may be too specific and therefore cannot detect antigens across different species, (3) not preferable for the antigens with low expression.

Apart from these considerations, some precautions should be taken into account when choosing primary and secondary antibodies. Primary antibody host that is the same as the tissue being studied should be avoided because the conjugated secondary antibody against the primary antibody may generate high background signals due to potential cross-reactivity (e.g., primary antibody against a rat protein should not be raised from rat or mouse). Instead, the secondary antibodies host should be the same species as the primary antibody being used. Also, researchers should pay attention to different forms of secondary antibodies, which can be either whole immunoglobulin G, F(ab’)₂ fragments, and Fab fragments. The whole immunoglobulin G is suitable for most IHC experiments and is most cost-effective, whereas F(ab’)₂ and Fab fragments can provide better signal-to-noise ratios and sensitivity due to their smaller size and faster diffusion through tissues. Sometimes, pAbs are preadsorbed with serum from various species in order to significantly reduce the cross-reactivity, which is a recommended choice.