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

Abstract

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 antibodies 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 immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope 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, CD79αcy+ B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+ neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+ astrocytes (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.

Phenotypic characterisation of cell populations in the brains of horses experimentally infected with West Nile virus

“…The following antibodies were used for targeting equine neural and inflammatory cells in FFPE tissue as described previously : polyclonal rabbit anti‐human CD3 + T cell antibody (A0452), monoclonal mouse anti‐swine glial fibrillary acid protein (GFAP) astrocyte antibody (clone 5C10), polyclonal rabbit anti‐ionised calcium binding adapter molecule‐1 (Iba‐1) microglia/macrophage antibody, monoclonal mouse anti‐human CD79αcy + B cell antibody (clone HM57), and monoclonal mouse anti‐human macrophage/granulocyte antibody (clone MAC387). Equine‐specific, monoclonal mouse anti‐equine CD4 + (clone HB61A) and monoclonal mouse anti‐equine CD8 + (clone HT14A) were used for staining T lymphocyte subpopulations in FFT.…”

“…Analysis of the inflammatory response within the brain of rodents has increased our insight to the immunological response to WNV meningoencephalomyelitis; however, similar quantitative statistical analysis of phenotyped cellular responses using cellular markers has not been performed in horses. In a previous study, we developed a cassette of antibodies that would stain resident CNS cell populations and peripheral blood mononuclear cells in formalin‐fixed paraffin‐embedded (FFPE) tissues . Techniques involving FFPE tissues are important to develop for diseases such as WNV since working with live virus infected fresh tissue requires high containment facilities.…”

“…Inflammatory cells are a limited component of CNS pathology overall for herpesvirus and for disease with inflammatory components phenotypic analysis of the CNS is limited. Based on a limited evaluation of WNV infected horses in a previous study , we hypothesised that acute inflammation in the brains of healthy mature horses undergoing grave intrathecal WNV challenge would respond locally with microglia proliferation and a comparatively mixed T cell response. Here, we quantify populations of infiltrating peripheral blood cells and resident cells in WNV experimentally infected brains of horses at the onset of neurological signs.…”

Acute Leukemia in Horses

“…Protocols for CD3, CD20, and CD79a IHC were previously established and yielded expected results with control tissues. 32 Iba-1/AIF-1 is considered a pan-macrophage marker in mice, 26 the antibody to Iab-1/AIF-1 bound to equine microglial cells 13 and labeled cells with macrophage and dendritic cell morphology in normal canine lymph node and equine bone marrow (data not shown). In addition, Iba-1/AIF-1 was also detected in rare lymphocytes in the paracortical T-cell–rich region of dog lymph node sections and in some blasts in equine bone marrow.…”

“…16,32 Additional antibodies tested were directed against ionized calciumbinding adapter molecule-1/allograft inflammatory factor-1 (Iba-1/AIF-1), CD34, myeloperoxidase (MPO), CD172a, CD163, and equine granulocytes, as summarized in Table 1. Antibody to Iba-1/AIF-1 has been reported to label all macrophage populations and spermatids in mice, 26 phagocytic macrophages and microglia in horse brain, 13 and neoplastic myelomonocytic cells and B lymphocytes in horse bone marrow. 5 Molecule CD34 is expressed by hematopoietic precursor cells and nonhematopoietic stem cells.…”