Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue
Gretchen H Delcambre1, Junjie Liu2, Jenna M Herrington2
1Department of Biomedical Sciencess/College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Insights
This study developed new antibody protocols for equine infectious encephalitides research. These methods improve the characterization of immune cells in horse central nervous system tissues.
Area of Science:
- Veterinary Pathology
- Immunohistochemistry
- Equine Neurology
Background:
- Limited reagents hinder detailed analysis of equine infectious encephalitides' cellular responses in the central nervous system (CNS).
- Characterizing peripheral and resident CNS cell populations is crucial for understanding neuroinflammation in horses.
- Existing methods lack specificity for formalin-fixed, paraffin-embedded (FFPE) equine tissues.
Purpose of the Study:
- To identify and optimize antibodies for immunohistochemistry (IHC) in equine CNS tissues.
- To establish reliable protocols for phenotypic characterization of cellular responses in equine neuroinflammatory diseases.
- To enable detailed investigation of immunopathology in horses.
Main Methods:
- Screened various commercial and species-cross-reactive antibodies for manual IHC.
- Optimized antigen retrieval (boiling in low pH citrate buffer), blocking, and detection systems for FFPE equine tissues.
- Tested multiple antibody clones and protocols, including monoclonal (mAb) and polyclonal (pAb) antibodies.
Main Results:
- Identified effective mAbs and pAbs for equine T-lymphocytes (CD3+), B-lymphocytes (CD79αcy+), macrophages (MAC387), neurons (NF-H+), microglia/macrophages (Iba-1+), and astrocytes (GFAP+).
- Human and swine-derived antibodies showed high reactivity with equine FFPE tissue targets.
- Optimized IHC protocols were developed for each validated antibody.
Conclusions:
- A validated set of antibodies and optimized IHC protocols are now available for equine neuroinflammatory disease research.
- These reagents facilitate detailed immunopathological analysis of equine CNS tissues.
- The findings address the need for specific reagents in equine neuropathology.
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.
