This article details a protocol for immunohistochemistry to detect rabies virus antigens in fixed mouse brain tissue. The method involves several steps including deparaffinization, rehydration, and antibody incubation to visualize the presence of the virus.
Begin with a slide carrying a formalin-fixed, paraffin-embedded rabies virus-infected mouse brain tissue section. The fixed section retains viral antigens via protein cross-linking.
Treat the slide with a xylene solution to remove the paraffin.
Immerse the slide in decreasing ethanol concentrations to rehydrate the tissue, making it compatible with aqueous solutions.
Introduce a protease-containing buffer to disrupt protein cross-links, improving antigen accessibility.
Submerge in hydrogen peroxide to block endogenous peroxidases, and apply a blocking solution to prevent non-specific interactions.
Introduce primary antibodies specific to the rabies antigen, forming antigen-antibody complexes.
Add biotinylated secondary antibodies, which bind to the primary antibodies. Incubate with streptavidin-conjugated peroxidase enzymes, which interact with biotin.
Introduce a peroxidase substrate, producing a magenta-red precipitate.
Counterstain the tissue with a nuclear stain, followed by a bluing agent, staining the nuclei blue.
The immunohistochemistry image reveals magenta-red spots in the cytoplasm, confirming the presence of rabies antigens in the tissue section.
Using a microtome, make a 5-micrometer paraffin section. Float the section on a water bath at 38 degrees Celsius, and collect it onto glass slides. Label the slides with a reagent-resistant pen. Place the slides onto a tray, and melt in an oven at 55 to 60 degrees Celsius for one hour.
Then, remove the slides from the oven, and immediately deparaffinize them using three consecutive xylene rinses of five minutes each. After this, rehydrate the sections on the slide by sequential immersions in decreasing dilutions of ethanol to deionized water, as outlined in the text protocol.
Treat the slides with the protease for 30 minutes for proteolytic antigen retrieval, then, rinse the slides in PBST for 10 minutes, and treat them with 3% hydrogen peroxide for 10 minutes. After this, wash the slides again with PBST for 10 minutes.
To begin, remove one slide from the buffer, making sure to only handle one slide at a time, while the others remain submerged, and use a paper towel to blot off excess buffer from around the tissue section. Place the slides into a humidity chamber, and incubate with normal goat serum at room temperature for 15 minutes.
Next, incubate the slides in the humidity chamber with the optimal predetermined dilution primary anti-rabies antibody and negative control antibodies at room temperature for 60 minutes with no washes in between. After this, wash the slides with PBST for 10 minutes, and incubate in a humidity chamber with the biotinylated antibody at room temperature for 15 minutes.
Wash the slides again with PBST for 10 minutes. Incubate the slides in a humidity chamber with streptavidin-HRP complex at room temperature for 15 minutes, and wash the PBST for 10 minutes. Then, incubate with AEC in a humidity chamber at room temperature for 10 minutes. Wash the slides in deionized water for 10 minutes and counterstain with Gill's hematoxylin diluted 1 to 2 with deionized water for two minutes.
After this, rinse off the excess hematoxylin by dip-rinsing the slides in deionized water. Rinse the slides in Scott's tap water for 30 seconds, and wash in deionized water for 10 minutes. Remove the slides one at a time and mount them with water-soluble mounting medium. Then, use a light microscope to read the slides.
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