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

Department of Biomedical Sciencess/ College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States
Department of Infectious Diseases and Pathology/ College of Veterinary Medicine, University of Florida, Gainesville, Florida, United States
Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida, United States
DOI
10.7287/peerj.preprints.1641v1
Subject Areas
Veterinary Medicine, Infectious Diseases, Pathology, Histology
Keywords
Immunohistochemistry, equine, neuropathology, leukocytes, neuroglia
Copyright
© 2016 Delcambre et al.
Licence
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
Cite this article
Delcambre GH, Liu J, Herrington JM, Vallario K, Long MT. 2016. ­Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue. PeerJ PrePrints 4:e1641v1

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.

Author Comment

This is a preprint submission to PeerJ Preprints.