Immunofluorescent staining of rabies virus antigen in formalin-fixed tissue after treatment with trypsin

Formalin-fixed central nervous system tissue from clinically rabid animals was treated with 0.25% trypsin and tested for the presence of rabies virus antigen by direct immunofluorescent (IF) staining. The results were comparable with those obtained from direct IF staining of acetone-fixed standard smears or fresh frozen-cut sections. Experiments were conducted using coded brain specimens (classified as IF-negative, weakly positive, or strongly positive) and showed a specificity of 100% for sections and 92% for smears; the latter figure was subsequently improved by modifying the preparation technique. The specificity of the technique was checked by standard virus neutralization of the conjugate, and by known antibody neutralization of the virus antigen in the specimens. The optimal duration for the trypsin digestion was found to be a minimum of 60 minutes at 37 degrees C or 120 minutes at 4 degrees C. The tissues could be held in buffered formalin for between 3 days and 7 weeks with no apparent difference in the results. Satisfactory concentrations of formalin were 0.125% or 0.25%. Trypsin was found to have no effect on non-formalinized tissues, with the exception that softening occurred making tissues harder to cut and process.The results suggest that trypsinization of formalin-fixed tissue is a valid procedure for the preparation of tissues for IF examination, which would be useful in cases where the current standard techniques cannot be used. However, further evaluation of the method is still required.