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.

Keywords: Equine; Immunohistochemistry; Leukocytes; Neuroglia; Neuropathology.

Figure 1. IHC of CD3⁺ T lymphocytes and CD79⁺ B lymphocytes in euqine tissues.

For the detection of lymphocytes, (A, C) equine lymph node cortex and (B, D) WNV infected equine brain incubated with (A, B) CD3⁺ T lymphocyte primary antibody (pAb A0452; Dako, Glostrup, Denmark) for 60 minutes at 37 °C and detected by Vectastain® ABC Kit, or incubated with (C, D) CD79αcy⁺ B lymphocyte primary antibody (mAb HM57, Dako) for 90 minutes at 37 °C and detected by Novolink™ Polymer Detection System. Vector NovaRED Peroxidase Substrate chromogen and hematoxylin couterstain. Bar, 50 μm.

Figure 2. IHC of MAC387⁺ and Iba-1⁺ macrophages and microglia in equine tissues.

For the detection of macrophage/microglial lineage cells, (A) equine thymus and (B) WNV infected equine brain were incubated with MAC387⁺ primary antibody (mAb MAC387; Leica, Wetzlar, Germany) for 60 minutes at 37 °C and detected by Vectastain® ABC Kit. Additionally, (C) normal (arrows) and (D) WNV infected horse brain were incubated with microglia/macrophage primary antibody (pAb Iba-1; Wako, Neuss, Germany) for 60 minutes at 37 °C and detected by Novolink™ Polymer Detection System. Vector NovaRED Peroxidase Substrate chromogen and hematoxylin couterstain. Bar, 50 μm.

Figure 3. IHC of GFAP⁺ astrocytes in WNV infected equine brain.

GFAP⁺ astrocytes in WNV infected equine brain (A) near blood vessels and (B) at the glial limitans. IHC. Bar, 50 μm.

Figure 4. IHC of NF-H⁺ neurons in equine brain.

Normal equine brain incubated with NF-H⁺ neuron primary antibody (mAb NAP4; EnCor Biotechnology, Gainesville, FL, USA), for 60 minutes at 37 °C and detected by Novolink™ Polymer Detection System. Vector NovaRED Peroxidase Substrate chromogen and hematoxylin couterstain. Bar, 50 μm.