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Home / Equine Research Bank / PeerJ / Immunohistochemistry for the detection of neural and inflamm...
PeerJ2016; 4; e1601; doi: 10.7717/peerj.1601

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.
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Publication Date: 2016-01-25 PubMed ID: 26855862PubMed Central: PMC4741088DOI: 10.7717/peerj.1601Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research paper presents a study aimed at identifying antibodies for investigating the immunopathology of infectious diseases in horses’ central nervous system (CNS) tissues.

Research Context and Objectives

  • Previous research on equine infectious encephalitides had faced limitations in characterizing cellular responses given the limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE).
  • This study aimed to identify a set of antibodies to fill this gap and aid in the investigation of the immunopathology of infectious CNS diseases in horses.

Research Methodology

  • The researchers began by screening numerous commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC).
  • They tested several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems during ICC protocol development.
  • The study found that boiling slides in a low pH, citrate-based buffer solution in a double-boiler system provided the most consistent results for epitope retrieval.

Findings and Outcomes

  • Other methods like pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often led to tissue disruption or no reactivity.
  • The study then determined optimal blocking reagents and concentrations for working with each antibody.
  • As a result of this work, they were able to identify a set of monoclonal antibodies (mAb) and polyclonal antibodies (pAb) for a range of cell types, including T-lymphocytes, B-lymphocytes, macrophages, neurons, microglia/macrophage, and astrocytes.
  • These monoclonal and polyclonal antibodies, derived from human and swine antigens, were successful at binding equine tissue targets in paraffin-embedded tissues.
  • The researchers then provided individual, optimized protocols for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

Significance and Implications

  • This study marks an advancement in veterinary research, potentially improving the ability to diagnose and understand diseases that affect horses’ nervous systems.
  • It validates the use of certain reagents for analyzing equine neuroinflammatory diseases, also making it easier for future studies to work around the limitations that this research was initially set up to address.

Cite This Article

APA
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, 4, e1601. https://doi.org/10.7717/peerj.1601

Publication

PeerJ
ISSN: 2167-8359
NlmUniqueID: 101603425
Country: United States
Language: English
Volume: 4
Pages: e1601
PII: e1601

Researcher Affiliations

Delcambre, Gretchen H
  • Department of Biomedical Sciencess/College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Liu, Junjie
  • Department of Infectious Diseases and Pathology/College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.
Herrington, Jenna M
  • Department of Infectious Diseases and Pathology/College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.
Vallario, Kelsey
  • Department of Infectious Diseases and Pathology/College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.
Long, Maureen T
  • Department of Infectious Diseases and Pathology/College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

Grant Funding

  • UL1 TR001427 / NCATS NIH HHS

Conflict of Interest Statement

The authors declare that they have no competing interests.

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Citations

This article has been cited 9 times.
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