Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

Tissues used for Immunohistochemistry and for diagnostic histopathology are routinely fixed in 10%phosphate buffer formalin. The detection of rabies virus antigen in brain tissue that has been fixed in formalin presents a unique challenge, and the IHC test has been found to be a sensitive and specific method for rabies virus antigen detection. The primary advantage of biopsy or other detection methods is that antigens can be detected in relation to tissue and cellular morphology.

With IHC, localization of rabies antigen in brain and non-brain tissues, provide additional information that could not be obtained through routine Hematoxylin and Eosin stain sections. Rabies diagnostic testing is important for the initiation of post-exposure prophylaxis in humans exposed to rabies virus. IHC is one of the most commonly performed assays on formalin-fixed tissues.

With specific antibodies, it can be used for the detection of respective pathogens. To begin this procedure, place three to five millimeter pieces of brain tissue, collected after necropsy, into a 10%buffered formalin solution for 24 to 72 hours. Record the approximate tissue weight, tissue type, and the volume of formalin.

Place the tissues in 70%ethanol for longer tissue storage after formalin fixation, and prior to processing. Following the specimen fixation, dissect the tissue to include the important brain areas, such as cross sections of the brainstem, the cerebellum, or the hippocampus. Each, cut to a thickness between three and five millimeters.

Place the tissues into processing cassettes. Process the tissue cassettes for paraffin wax infiltration. Embed them into paraffin blocks and section them on a microtome.

First set up the staining dish as outlined in Figure 1 of the text protocol. Fill each dish with 250 milliliters of the prepared solution. To prepare the AEC substrate stock solution, use a glass pipette to dissolve one 20 milligram tablet of AEC in 5 milliliters of N, N-Dimethylformamide.

To prepare the protease stock solution, dissolve 7 milligrams of the protease in 200 milliliters of PBS. To prepare the rinse buffer, add 100 milliliters of Tween 80 to 990 milliliters of PBS, and mix well to form a homogenous solution of PBT-T. Using a microtome, make a five micrometer paraffin section.

Load 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 3 consecutive xylene rinses of 5 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 PBS-T for 10 minutes, and treat them with 3%hydrogen peroxide for 10 minutes.

After this, wash the slides again with PBS-T 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 PBS-T for 10 minutes and incubate in a humidity chamber with the biotinylated antibody at room temperature for 15 minutes. Wash the slides again with PBS-T for 10 minutes.

Incubate the slides and the humidity chamber with strip divide and HRB complex at room temperature for 15 minutes and wash the PBS-T 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 Hematoxilyn, diluted one to two 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. In this study, an immunocytic chemistry test is demonstrated for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues. A representative immunocytic chemistry staining result of positive and negative control samples in different brain tissues is shown here, including the brainstem, the cerebellum and purkinje cells, and the hippocampus.

The magenta red staining, which indicates the color development by using AEC substrate against the blue counterstained background, is due to the reactivity of antibodies against the rabies antigen. A positive result in Immunohistochemistry corresponds to the magenta red staining in the tissue sections. The staining of cytoplasmic inclusions and granular inclusions of varying size are indicative of samples positive for RABV infections.

Samples are deemed negative if no specific red staining or only the blue background, due to hematoxylin, were observed. In addition to the positive staining, the distribution of inclusions could provide indirect quantification of levels of rabies antigen in the sample, which might correspond to the viral load of the tissue sample. Irrespective of the levels of distribution, any specific staining will classify the sample as positive for RABV antigen detection.

Tissues should be fixed adequately in formalin, and avoid freezing the tissues during this process. Rabies antigen is detected by IHC, then molecular techniques can be performed on paraffin sections to determine the Lyssavirus variant based on RNA sequence information. Reagents like xylene and formalin should be handled in fume hoods.

Care should be taken while handling AEC as it is a carcinogen.