This was followed by harvesting of brain tissues, which were placed in 10% Histopathology and immunohistochemical analysis of anti-p53 and anti-Bcl Jan 21, Histopathological and immunohistochemical comparison of the brain of human patients with Alzheimer's disease and the brain of aged dogs. Jan 25, Comparative pathology of many diseases that affect multiple species is .. Additionally in brain tissue, this antibody resulted in non-specific.
immunohistochemistry Brain histopathology and
Nuclear Extract Preparation Mike A. Stable Lines Mike A. Successfully culturing Schwann cells from adult peripheral nerve A. Co-culturing neurons and microglia using tissue culture inserts Tsirka Lab, Stony Brook. Brain Atlas of the Rhesus monkey BrainMuseum. Brain Atlas of the Squirrel monkey BrainMuseum. Alex Becker Harvard Medical School. The Human Brain Atlas. Neuroanatomy Structures University of Debrecen. A Brief Tutorial University of Debrecen. Neuroanatomy Tutorial University of Utah.
Neuroanatomy Atlas Saint Joseph's University. Atlas Images including Neuroanatomy University of Michigan. Blocking reagents were removed without rinsing before adding primary antibody. Primary antibodies tested targeted cell populations of astrocytes, microglia, neurons, T lymphocytes, B lymphocytes, and macrophages Table 1. Two-fold serial dilutions of each antibody were tested to determine an optimal staining range.
If the signal was weak to absent or background staining was present, additional dilution tests were performed until optimal staining was achieved. Negative controls for each primary antibody consisted of either an isotype-matched negative primary control MCA, AbD Serotec, Kidlington, UK for monoclonal antibodies or rabbit serum for polyclonal antibodies. Of the reagents tested, low pH citrate based solutions resulted in superior staining Table 2.
Various issues were encountered with the method of applying heat. Pressure cooking and microwaving resulted in limited to no staining or uneven staining, respectively. In addition, tissue disruption was minimal with the double boiling method. There was no difference between commercial and lab diluted reagents. Multiple combinations of IHC antibody diluents and non-specific protein blocking reagents were tested. No difference in staining intensity was noted between of either diluent used.
Although this antibody intensely stained the germinal centers of FFPE lymph nodes, cortical staining was also noted. Additionally in brain tissue, this antibody resulted in non-specific background staining, which could not be resolved.
Multiple macrophage-targeting antibodies were investigated. This marker positively stained control hepatic and thymic macrophages Fig. Based on cell morphology, polymorphonuclear and mononuclear cells also stained positively due to lack macrophage-specificity of the antibody. No reactivity was noted in normal equine brain. To characterize reactive gliosis, antibodies against both microglial and astrocytic markers were tested. Staining intensity was higher in WNV-infected brains Fig. Astrocyte populations were identified with antibody against an intermediate filament, glial fibrillary acid protein GFAP , specific to astrocytes in CNS tissues.
This antibody reacted strongly with astrocytes in both normal and infected brains with distinct astrocytic processes notable. Three antibodies that target neurofilament heavy-chain proteins NF-H of neuronal axons successfully stained equine brain tissue. Successful manual IHC protocols for these antibodies in equine neural tissue were identified Table 2.
Formalin-fixed, paraffin embedded tissues are commonly archived for histological examination. Formalin fixation and paraffin embedding samples retains tissue architecture and deactivates any potential infectious agents Cantile et al.
This result has been described in other species Beckstead, ; Bilzer et al. Archiving tissues in both FFPE and fresh, frozen tissue formats is recommended when investigating epitopes that may be affected by fixation. After successful staining of control tissues, normal and pathologic brain tissue samples were then tested alongside positive and negative tissue controls. Pathologic tissue samples included WNV-infected brain, which contains reactive gliosis and perivascular cuffing of inflammatory cells Cantile et al.
Following this workflow of tissue testing aided in the identification of successful antibody reactivity. Several hurdles must be overcome to optimize the interaction of antibodies with their intended targets. These unwanted binding sites must be blocked.
In this study a variety of reagents and methods were tested for each individual manual IHC protocol. A base set of reliable solutions and techniques for testing antibodies was identified in this study.
However, with meticulous tailoring of each antibody protocol, our results contained a variety of reagents that were ultimately selected for each staining procedure. Evaluation of cross-species reactivity of commercially available antibodies, particularly CD antigens, has been largely performed in equine tissues prepared for whole-cell analysis like flow-cytometry Johne et al.
Large screenings of non-equine derived antibodies have often resulted in limited identification of equine reactive reagents Ibrahim et al. Of the 26 antibodies in this study, six antibodies successfully reacted with FFPE equine tissues. All six antibodies were derived from non-equine antigens. Their reactivity to equine tissue is likely due to conserved epitope targets and the intracellular location of the antigen peptides Jones et al.
However, because these antigens have intracellular origins, the host i. Commercial macrophage antibodies are non-specific to macrophages and often cross react with monocytes, granulocytes, dendritic cells, and fibroblasts Johne et al. Multiple macrophage-directed antibodies with different target antigens were tested so that a distinction could be made between tissue macrophage populations and cells that may cross-react with those antibodies.
RAM11 Dako, Glostrup, Denmark recognizes an uncharacterized, cytoplasmic antigen specific to rabbit macrophages. AM-3K TransGenic, Strasborg, France anti-macrophage antibody raised against human alveolar macrophage antigen recognizes cytoplasmic membrane epitopes.
None of these three antibodies were successfully reactive in FFPE brain or lymphoid tissues. Microglia, which are of a monocytic lineage, were identified with anti-Ibaantibody Wako, Neuss, Germany. This antibody recognizes an intracellular, calcium binding protein basally expressed by both microglia and macrophages, and upregulated in microglia following injury.
Cell morphology, specifically microglial processes, may be used to manually separate microglia and macrophage populations. Additionally, using MHC as a marker for microglial cells in an inflammatory disease would also identify all peripheral and CNS cells that have MHC upregulated due to this biological process.
Regarding detection of neurons, it must be recognized that NF-H proteins are concentrated within axons and are weakly present within cell bodies. Changes in staining intensity within cell bodies and dendrites with NF-H antibodies, such as NAP4 that targets phosphorylated NF-H proteins, are useful for identifying neuropathology Shaw et al. For quantification of neuronal populations, an antibody specific to neuronal nuclei should be used.
This study successfully identified a collection of protocols for the characterization of cell populations in the histopathology of encephalitic diseases in equine CNS tissues. Useful application of these antibodies was supported with the characterization of neuropathology in diseased horses with clinical and experimental West Nile encephalitis, clinical equine protozoal myeloencephalitis, and, preliminarily, clinical Eastern equine encephalitis.
Western blot analysis utilizing these antibodies should next be performed to support the specificity of their reactivity in the horse. We would like to thank Dr. Gerry Shaw and Dr. Jake Streit for their consultation and loan of antibodies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests The authors declare that they have no competing interests.
Author Contributions Gretchen H. Junjie Liu performed the experiments, analyzed the data, reviewed drafts of the paper. Herrington performed the experiments. Kelsey Vallario performed the experiments. Animal Ethics The following information was supplied relating to ethical approvals i.
Data Deposition The following information was supplied regarding data availability:. National Center for Biotechnology Information , U. Journal List PeerJ v. Published online Jan Delcambre , 1 Junjie Liu , 2 Jenna M. Herrington , 2 Kelsey Vallario , 2 and Maureen T. Author information Article notes Copyright and License information Disclaimer. Received Oct 2; Accepted Dec 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.
This article has been cited by other articles in PMC. 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. Immunohistochemistry, Equine, Neuropathology, Leukocytes, Neuroglia. Introduction Comparative pathology of many diseases that affect multiple species is hindered by the lack of species-specific reagents.
Materials and Methods Tissue samples Immunohistochemistry protocols were developed on diseased and normal horse tissues. Antigen unmasking Three methods of heating slides for investigating heat induced epitope retrieval HIER effectiveness included using a pressure cooker, microwave, and a double boiler.
Non-specific protein blocking Non-specific blocking techniques included four commercial reagents and one lab prepared solution. Primary antibodies Primary antibodies tested targeted cell populations of astrocytes, microglia, neurons, T lymphocytes, B lymphocytes, and macrophages Table 1. Table 1 Antibodies tested for immunohistochemical reactivity in equine tissues. Open in a separate window. Results Antigen unmasking Of the reagents tested, low pH citrate based solutions resulted in superior staining Table 2.
Table 2 Selected protocols for immunohistochemical visualization of resident and infiltrative cells in normal and WNV-diseased equine CNS tissues.
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Key words: Encephalitozoon cuniculi; immunohistochemistry; rabbits; real-time polymerase Histopathological examination was performed on brain, heart. Mouse-brain slice stained by Immunohistochemistry. Immunohistochemistry (IHC ) is the most common application of immunostaining. It involves the . The diversity of IHC markers used in diagnostic surgical pathology is substantial. including immunohistochemistry and real-time polymerase chain reaction (PCR) for Histopathological examination was performed on brain, heart, lungs.