Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antiboies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various, species for manual immun H ohistochemistry (I HC) screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization detection systems were tested during IH C protocol development. Boiling of slides in a lowpH, citrate-based buffer solution in a double-boiler system was most consistent for epito pe retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3 (pAb A0452, Dako) T-lymphocytes, CD791acy B-lymphocytes (mAb HM57, Dako),' macrophages (mAb MAC387, Leica), NF-H+ neurons (mAb NAP4, EnCor Biotechnology), micruglia/macruPhage (pAb 113a-1, Wako), and GFAP+ astrucYtes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.