An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Formalin fixation of tissues

1. Place 3-5 mm brain tissues collected after necropsy into 10% phosphate-buffered formalin solution (1:20 to 1:50 tissue/formalin ratio) for 24-72 h.
   CAUTION: Formalin is a toxic fixative.
2. Record the approximate tissue weight (size), tissue type (brain areas), and volume of formalin. It is important for laboratories to document and maintain records on fixation times.
3. For longer tissue storage after formalin fixation and prior to processing, place the tissue in 70% ethanol.

2. Tissue processing

1. Following the specimen fixation, dissect the tissue to include the important brain areas e.g., cross sections of the brainstem, cerebellum (3 lobes), or the hippocampus (both hippocampi) each cut 3 to 5 mm thick and placed into processing cassettes.
2. Process the tissue cassettes for paraffin wax infiltration, embedded into paraffin blocks and sectioned (3 to 6 µm) on a microtome.

3. Preparation of Materials / Staining dishes

1. Set up the staining dish (Table of Materials) as shown in Figure 1. Fill each dish with 250 mL of the solution.
2. Preparation of the 3-Amino-9-ethylcarbizole (AEC) substrate stock solution
   1. Dissolve one 20 mg tablet of 3-amino 9-ethylcarbazole (AEC) into 5 mL of N,N, dimethylformamide using a glass pipette.        
      CAUTION: AEC is a carcinogen.
3. Preparation of the protease (e.g., Pronase) stock solution for the antigen retrieval
   1. Dissolve 7 mg of the protease in 200 mL of phosphate-buffered saline (PBS).
4. Preparation of the rinse buffer PBS-T
   1. Add 10 mL of Tween 80 to 990 mL of PBS. Mix it well to form a homogenous solution.

4. Deparaffinization and tissue rehydration

1. Make a 5 µm paraffin section using a microtome, float it on a water bath at 38 ˚C, and collect it onto glass slides. Label the slides with a reagent-resistant pen/marker.
2. Place the slides onto a tray and melt in a 55-60 ˚C oven for 1 hour. Do not raise the temperature above 60 ˚C as it can destroy the viral antigen.
3. Remove slides from the oven and immediately deparaffinize in 3 consecutive xylene rinses of 5 min each in dishes 1, 2, and 3.
4. Rehydrate the sections on the slide by sequential immersions in decreasing dilution of ethanol to deionized water: (4 through 11 is a dip rinse) dish 4: xylene/100% ethanol (1:1); dish 5: 100% ethanol; dish 6: 100% ethanol; dish 7: 95% ethanol; dish 8: 95% ethanol; dish 9: 80% ethanol; dish 10: 70% ethanol; dish 11: deionized water (Figure 1. Dish set-up).
   CAUTION: Xylene is a hazardous chemical and work should be conducted in a fume hood.

5. Proteolytic antigen retrieval

1. Treat slides with the protease (2.5 µg/mL of PBS) for 30 min for proteolytic antigen retrieval in dish 12.
2. Then rinse in PBS-T for 10 min (dish 13).
3. Treat with 3% hydrogen peroxide for 10 min (dish 14).
4. Again, wash with PBS-T for 10 min (dish 15).

6. Staining procedure

1. Handle slides one at a time keeping the remaining slides submerged in the buffer (do not remove the whole slide holder – keep slides wet). Remove one slide and blot off excess buffer (using a paper towel) from around the tissue section being careful not to disturb the tissue section. Incubate slides in a humidity chamber, made by placing moistened paper towels, on the lab bench top at room temperature with normal goat serum (blocking) for 15 min.
2. Incubate with the optimal pre-determined dilution primary anti-rabies antibody (1:250 dilution of mouse anti-rabies serum, unpublished) (positive control) and negative control antibodies at room temperature same as above (step 6.1.) for 60 min with no washes in between.
3. After 60 min, wash with PBS-T for 10 min (dish 16).
4. Incubate with the biotinylated antibody (species-specific) in a humidity chamber at room temperature for 15 min (handling same as step 6.1.).
5. Wash with PBS-T for 10 min (dish 16).
6. Incubate with Streptavidin- horseradish peroxidase (HRP) complex in a humidity chamber at room temperature for 15 min (handling same as 6.1.).
7. Wash with PBS-T for 10 min (dish 16).
8. Incubate with peroxidase substrate, amino-ethylcarbazole (AEC), in a humidity chamber at room temperature for 10 min. Make AEC just prior to use. To do so, add 1 mL of AEC stock solution to 14 mL of 0.1M acetate buffer, pH 5.2. Add 0.15 mL of 3% peroxide, H₂O₂. Filter the mixture just before use (0.45 µm filter).
   NOTE: The working solution of AEC is only stable for 2-3 hours. The AEC stock solution can be stored in the refrigerator for longer periods.
9. Wash in deionized water for 10 min (dish 17)
10. Counterstained with Gill's Hematoxylin diluted 1:2 with deionized water for 2 min (dish 18).
11. Rinse off excess Hematoxylin with deionized water dip rinse (dishes 19 and 20).
12. Rinse in Scott's Tap water for 30 s (bluing solution) dish 21.
13. Wash in deionized water for 10 min (dish 22)
14. Remove slides one at a time – mount with a water-soluble mounting medium.
15. Read slides on a light microscope.