Name: detection of abnormal prion protein by immunohistochemistry
Uploaded: 2023-05-05T14:20:00
Description: Watch this Scientific Journal Video about Detection of Abnormal Prion Protein by Immunohistochemistry at JoVE.com

This article was funded by the Project POCI-01-0145-FEDER-029947 &#34;Chronic wasting disease risk assessment in Portugal&#34; supported by FCT (Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia) - FEDER -Balc&#227;o2020. Also, the authors of the research unit CECAV received funding from the FCT, under the project UIDB/CVT/0772/2020.We thank Bruce C. Campbell, Research Director (retired), Western Regional Research Center, USDA, for his assistance.

The IHC procedure described herein is a component of the research project FAIRJ-CT98-7021, &#34;The establishment of a European network for the surveillance of ruminant TSE and the standardization and harmonization of the process and criteria for the identification of suspect cases&#34;. It follows the diagnostic criteria defined by the World Organization for Animal Health, WOAH (previously named the Office International des &#201;pizooties, OIE)1 and European Reference Laboratory for animal TSEs<...

<ol>
	<li>. WOAH, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Online Access. Chapter 3.4.5.-Bovine Spongiform Encephalopathy (Version May 2021) and Chapter 3.8.11. - Scrapie (Version May 2022) Available from: <a target="_blank" href="https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-manual-online-access/">https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-manual-online-access/</a> (2022)</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+methods+of+immunohistochemistry.">Principles and methods of immunohistochemistry.</a> Methods in Molecular Biology. 1641, 115-128 (2017).</li><li>Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M., Oliver, C., Jamur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-Induced+antigen+retrieval+for+immunohistochemical+reactions+in+routinely+processed+paraffin+sections.+Immunocytochemical+Methods+and+Protocols.">Heat-Induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections. Immunocytochemical Methods and Protocols.</a> Methods in Molecular Biology. 588, 103-119 (2010).</li><li>Duraiyan, J., Govindarajan, R., Kaliyappan, K., Palanisamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+immunohistochemistry).">Applications of immunohistochemistry).</a> Journal Pharmacy Bioallied Sciences. 4, 307-309 (2012).</li><li>Orge, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathology+of+Animal+Prion+Diseases.">Neuropathology of Animal Prion Diseases.</a> Biomolecules. 11 (3), 466 (2021).</li><li>. Biosafety in Microbiological and Biomedical Laboratories. 6th Edition Revised June 2020 Available from: <a target="_blank" href="https://www.cdc.gov/labs/pdf/SF_19_308133-A_BMBL6_00-BOOK-WEB-final-3.pdf">https://www.cdc.gov/labs/pdf/SF_19_308133-A_BMBL6_00-BOOK-WEB-final-3.pdf</a> (2022)</li><li>APHA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation,+tissue+processing,+histology+and+immunohistochemistry+procedures+for+diagnosis+of+animal+TSE+(BSE,+scrapie,+atypical+scrapie,+CWD).+Histo+&amp;+IHC+protocols+for+TSE+diagnosis_Rev_Jan2019.pdf.">Fixation, tissue processing, histology and immunohistochemistry procedures for diagnosis of animal TSE (BSE, scrapie, atypical scrapie, CWD). Histo &amp; IHC protocols for TSE diagnosis_Rev_Jan2019.pdf.</a> TSEglobalNet - International Reference Laboratory for TSE. , (2022).</li><li>Sample requirements for TSE testing and confirmation. Version 1.0. TSE EURL Available from: <a target="_blank" href="https://www.izsplv.it/it/istituto/212-centri-eccellenza/laboratori-internazionali-riferimento/422-eurl_tses.html">https://www.izsplv.it/it/istituto/212-centri-eccellenza/laboratori-internazionali-riferimento/422-eurl_tses.html</a> (2019)</li><li>TSE EU Reference Laboratory Guidelines for the detection of Chronic Wasting Disease in cervids. Version 1.0. TSE EURL Available from: <a target="_blank" href="https://www.izsplv.it/it/istituto/212-centri-eccellenza/laboratori-internazionali-riferimento/422-eurl_tses.html">https://www.izsplv.it/it/istituto/212-centri-eccellenza/laboratori-internazionali-riferimento/422-eurl_tses.html</a> (2019)</li><li>Machado, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TSE+Monitoring+in+Wildlife+Epidemiology,+Transmission,+Diagnosis,+Genetics+and+Control.">TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control.</a> Wildlife Population Monitoring. IntechOpen. , (2019).</li><li>APHA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathology:+Confirmatory+diagnosis+of+transmissible+spongiform+encephalopathies+(TSEs)+in+cattle+and+small+ruminants.+Confirmatory+(Histo+&amp;+IHC)+diagnostic+criteria+Rev_Jan2019.pdf.">Neuropathology: Confirmatory diagnosis of transmissible spongiform encephalopathies (TSEs) in cattle and small ruminants. Confirmatory (Histo &amp; IHC) diagnostic criteria Rev_Jan2019.pdf.</a> TSEglobalNet - International Reference Laboratory for TSE. , (2019).</li><li>Ryder, S. J., Spencer, Y. I., Bellerby, P. J., March, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+detection+of+PrP+in+the+medulla+oblongata+of+sheep:+The+spectrum+of+staining+in+normal+and+scrapie-affected+sheep.">Immunohistochemical detection of PrP in the medulla oblongata of sheep: The spectrum of staining in normal and scrapie-affected sheep.</a> The Veterinary Record. 148 (1), 7-13 (2001).</li><li>Simmons, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+classical+bovine+spongiform+encephalopathy:+definition+and+progression+of+neural+PrP+immunolabeling+in+relation+to+diagnosis+and+disease+controls.">Experimental classical bovine spongiform encephalopathy: definition and progression of neural PrP immunolabeling in relation to diagnosis and disease controls.</a> Veterinary Pathology. 48 (5), 948-963 (2011).</li><li>Orge, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+H-type+BSE+in+Portugal.">Identification of H-type BSE in Portugal.</a> Prion. 9 (1), 22-28 (2015).</li><li>Orge, L., Simas, J. P., Fernandes, A. C., Ramos, M., Galo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarity+of+the+lesion+profile+of+BSE+in+Portuguese+cattle+to+that+described+in+British+cattle.">Similarity of the lesion profile of BSE in Portuguese cattle to that described in British cattle.</a> Veterinary Record. 147 (17), 486-488 (2000).</li><li>Pires, M. A., Travassos, F. S., G&#228;rtner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atlas+of+veterinary+pathology.">Atlas of veterinary pathology.</a> Biopathology. Lidel VII. 195 (6), 179-180 (2004).</li><li>Pires, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+protocols,+didactic+series.">Immunology protocols, didactic series.</a> Applied Sciences. , 357 (2010).</li></ol>

TSEs are potential zoonotic diseases. After the emergence of BSE in 1986 in the United Kingdom, Portugal became one of the European Union Member States with a higher incidence of this disease14,15. In order to control this disease, other TSEs that have emerged (classical and atypical scrapie, BSE variants, and currently the surveillance of chronic wasting disease in cervids), surveillance mechanisms were developed by the Directorate General of Food and Veterinary...

According to the prion hypothesis, the abnormal isoform (PrPSc) is the primary, or sole, component of the infectious agent in transmissible spongiform encephalopathies (TSEs). Confirmation for diagnosis of TSE relies on immunodetection of PrPSc by application of immunohistochemistry (IHC) protocols and/or western immunoblot methods (WB) of encephalon tissues1.
IHC is a method employing&#160;monoclonal or, in some cases, polyclonal antibodies (as primary antibodies) as a first step in immunostaining of specifically targeted antigens of interest located in cells of a tissue section. Any effective primary antibody-antigen binding is then visualized using secondary antibodies specific to the primary antibodies. These secondary antibodies are conjugated to enzymes, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Visualization is then achieved by adding a substrate to these enzymes, producing insoluble color products localized in areas where the primary antibodies are bound to the targeted antigens. Improved visualization can be achieved by counterstaining, wherein a dye is used to create a contrast between immunolabeled and un-immunolabeled tissue2.
With IHC using formalin-fixed paraffin-embedded tissues (FFPE), formalin fixation can nullify the effectiveness of primary antibodies due to cross-linking by formaldehyde and heating and dehydration during paraffin embedding. These change the conformation of proteins, destroying, denaturing, or masking the epitopes, thus diminishing or abrogating their detection3. As such, this requires antigen retrieval (AR). The AR techniques disrupt formaldehyde-related chemical group cross-linking in the antigenic molecules, thus restoring or unmasking the original antigen-protein conformation. This results in recovering the antibody-antigen (epitope) affinity for immunolabeling. The eventual efficacy of AR depends on the qualities of the targeted antigen and/or the primary antibody2.
Heat-induced antigen (epitope) retrieval (HIER) is one procedure of AR3 and is used routinely for PrPSc IHC detection, as described herein. IHC is essential for diagnosis and used in research laboratories to determine the tissue distribution of a pathology-associated antigen. It is widely used in diagnosing and researching cancer, neuroscience, and infectious diseases4, among others. For TSEs, IHC plays an important role in diagnosis and research for confirming and investigating PrPSc distribution in natural hosts and experimental models. IHC contributes to prion pathogenesis studies and the analysis of PrPSc deposition types and patterns, namely, in neural tissues5, to detect deviations from routinely described infections and identify putative new prion strains.
Because prions of bovine spongiform encephalopathy (BSE) can infect humans, certain laboratory protocols involved in the work with BSE may require the use of BSL-3 facilities and practices6. These include using a sealed secondary container to transport potential BSE-infected tissue samples within the institute and laboratory. It also includes designating containment areas and prion-dedicated equipment for BSE research and analysis whenever possible. This is done to prevent contamination outside the work area and provide a confined space since decontamination procedures become necessary.
Accordingly, the Laboratory of Pathology of INIAV follows recommended biosafety level-3 (BSL-3) facilities and practices6 to manage potential prion-infected samples of tissues from cattle, small ruminants, and cervids associated with the TSE surveillance.
Formalin-fixed and paraffin-embedded tissues included in TSE diagnostic or research procedures, especially in the central nervous system, can be potentially infectious. Hence, these fixed tissues must be treated with formic acid to reduce the infectivity of prions, if present, prior to tissue processing. This is performed by placing fixed, trimmed tissues (approximately 2-4 mm thickness) in a processing cassette. The cassette is then immersed in 98% formic acid (for 1 h). After immersion, the cassettes with tissues are washed in running tap water for 30 min, and returned to the fixative before further processing. If tissue sections are not treated before processing, post-microtomic tissue sections must be immersed in undiluted formic acid for at least 5 min before histological staining7. The IHC protocol for PrPSc includes a routine formic acid epitope-demasking step, also serving to inactivate prions7. After these prion inactivation steps, the resulting fixed tissues can then be processed at BSL-2 using standard BSL-2 practices.
The minimum tissue sampling requirement for TSE diagnosis in any animal included in TSE surveillance is collecting the brainstem (at the obex level). Additionally, for detecting atypical BSE and scrapie, it is advised that part of the cerebellum should also be collected1,8. For CWD diagnosis, both brainstem (obex) and retropharyngeal lymph nodes should be tested as PrPSc could be detected in lymphoid tissues with no detectable PrPSc in obex9, reviewed by Machado et al.10.
The obex portion of the brainstem includes diagnostic TSE target sites, namely, the dorsal vagal nucleus (DVN), solitary tract nucleus (STN) and spinal tract nucleus of the trigeminal nerve (V). These areas consistently present bilateral PrPSc accumulation, even in the early stages of BSE and classical scrapie. In clinical cases of advanced TSE, all the gray-matter areas within the brainstem show widespread PrPSc distribution11.
Before sectioning and processing, the brain samples are evaluated (Figure 1) to ascertain the level of autolysis and the presence of any tissue damage potentially compromising the suitability of the sample for IHC-based confirmatory diagnosis8. To validate the integrity of the preparative protocols and the analytical results, the TSE positive and negative tissue samples are included as controls in conjunction with the preparation of tissues from test cases in each assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg" /> 
Figure 1: The PrPSc IHC procedure. Representation showing the step-by-step sequence of PrPSc IHC procedure from deparaffinization of tissue sections to eventual immunostaining and detection (FFPE - Formalin-fixed paraffin-embedded; Mab - monoclonal antibody; DAB - 3,3&#39; diaminobenzidine). This figure was created in BioRender.com. <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absolute ethanol&#160;</td>
			<td>Labchem</td>
			<td>LB0507-9010</td>
			<td>Undituled</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Diluted 90%, 70% and 50% in distilled water</td>
		</tr>
		<tr>
			<td>Avidin-biotin complex and peroxidase 
			Vectastain Elite ABC kit Peroxidase</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>Prepare and gently mix 30 min before use according to kit instructions. Do not mix after standing.</td>
		</tr>
		<tr>
			<td>Biotinylated secondary antibody (Horse anti-mouse IgG H+L)&#160;</td>
			<td>Vector Laboratories</td>
			<td>BA-2000-1.5</td>
			<td>Dilute at 1/200 in TBS with 10% horse normal serum. Prepare the volume required depending on the number of sections.</td>
		</tr>
		<tr>
			<td>Chromogen Diaminobenzidine- DAB, substrate kit, Peroxidase&#160;</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>Prepare before use according to kit instructions. 
			Use 400 &#181;L of solution per section.</td>
		</tr>
		<tr>
			<td>DakoCytomation Pascal pressure chamber</td>
			<td>DAKO</td>
			<td>S2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ehrlich&#8217;s Hematoxylin:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Merck</td>
			<td>115938</td>
			<td>Dissolve 2 g of hematoxylin in 100 mL of absolute ethanol. Add 100 mL of distilled water, 10 mL of glacial acetic acid and 15 g of potassium alum with constant stirring. Add 100 mL of glycerin. The natural oxidation process takes 2 months, before use.</td>
		</tr>
		<tr>
			<td>Absolute ethanol&#160;</td>
			<td>Labchem</td>
			<td>LB0507-9010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid&#160;</td>
			<td>Merck</td>
			<td>101830</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium alum</td>
			<td>Merck</td>
			<td>1.01047.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>Merck</td>
			<td>1.04091.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Endogenous Peroxidase Block solution (3% concentration H2O2):</td>
			<td></td>
			<td></td>
			<td>40 mL Hydrogen peroxide (30% w/w) in 360 mL Methanol. 
			Prepare before use</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (30% w/w)</td>
			<td>Scharlau</td>
			<td>HI0136</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>322415-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid 98%</td>
			<td>Merck</td>
			<td>1.00264.1000</td>
			<td>Undiluted</td>
		</tr>
		<tr>
			<td>Microtome&#160;</td>
			<td>Shandon-AS325</td>
			<td>Microtome&#160;</td>
			<td>Shandon-AS325</td>
		</tr>
		<tr>
			<td>Mounting medium Entellan</td>
			<td>Merck</td>
			<td>107960</td>
			<td>Ready- to- use.</td>
		</tr>
		<tr>
			<td>Normal serum (20% ) block solution in TBS: 
			Horse normal serum</td>
			<td> 
			Gibco</td>
			<td> 
			16050-122&#160;</td>
			<td>Prepare final volume according to the number of sections in the assay 
			(200 &#181;L of solution per section).</td>
		</tr>
		<tr>
			<td>Primary antibody anti-PrP Mouse MAb 2G11&#160;</td>
			<td>BIORAD</td>
			<td>MCA2460</td>
			<td>PrP 146-R154R171182 
			Ovine including atypical scrapie, cervine, feline. Not suitable for bovine. 
			According to the number of sections in the assay (200 &#181;L of solution per section) and antibody dilution, prepare final volume in TBS supplemented with 10% of normal serum from the species the secondary antibody was raised in (horse normal serum) 
			Usual antibody dilution: MAb 2G11 1/100 but working dilution should be established in every new batch to get the concentration to give the strongest labelling with lowest background. For storage, freeze aliquot volumes of a minimum of 10 &#956;L into sterile microtubes. Defrost and use one aliquot at a time.&#160;</td>
		</tr>
		<tr>
			<td>Primary antibody anti-PrP Mouse MAb 12F10&#160;</td>
			<td>Cayman&#160; Chemical Company&#160;</td>
			<td>189710</td>
			<td>PrP142-160 
			Bovine, not suitable for ovine 
			Usual antibody dilution: 1/200 but working dilution should also be established. Prepare as MAb 2G11</td>
		</tr>
		<tr>
			<td>Shandon CoverplateTM chamber</td>
			<td>Thermo Scientific&#160;</td>
			<td>72110017</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Sequenza&#174; Immunstaining center&#160;</td>
			<td>Thermo Scientific</td>
			<td>73300001</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Sequenza&#174; Immunstaining slide rack</td>
			<td>Thermo Scientific</td>
			<td>73310017</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution Citrate Buffer (10 mM pH 6.1):</td>
			<td></td>
			<td></td>
			<td>2.55 g Tri-sodium citrate dihydrate&#160; and 0.255 g Citric acid in one litre purified water. 
			Adjust pH of working solution to 6.1 using 10 mM citric acid solution (1.05 g citric acid in 500 mL purified water) 
			Prepare on assay day.&#160;</td>
		</tr>
		<tr>
			<td>Tri-sodium citrate dihydrate</td>
			<td>Sigma-aldrich&#160;</td>
			<td>S4641-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma Aldrich</td>
			<td>C0759</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining jar and basket</td>
			<td>Deltalab&#160;</td>
			<td>19360</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>19361</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus microscope slides</td>
			<td>VWR</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Buffered Saline solution (TBS) (50 mM TRIZMA BASE; 0.8% NaCI; pH 7.6):</td>
			<td></td>
			<td></td>
			<td>10xTBS (stock solution 0.5 M TRIZMA BASE; 8% NaCI; pH 7.6): 
			TRIZMA BASE 60,57 g and NaCl 80 g in 800 mL purified water. Adjust pH of stock solution using Hydrochloric acid 37% and final volume to one litre with purified water (keep 5&#177; 3 &#176;C until 2 months) 
			Dilute TBS stock solution 1/10 on assay day.&#160;</td>
		</tr>
		<tr>
			<td>TRIZMA BASE</td>
			<td>Sigma Aldrich</td>
			<td>T6066-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Merck</td>
			<td>106404</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Panreac Applied Chem ITW reagents</td>
			<td>251769</td>
			<td>Undiluted</td>
		</tr>
	</tbody>
</table>

detection of abnormal prion protein by immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Given that non-specific target immunolabeling is possible, it is important to ascertain the level of non-specific immunolabeling in known TSE-negative control animals. This is an important step to properly interpret specific PrPSc immunolabeling7 (Figure 2). Non-target labeling by anti-PrP antibodies has been noted to occur as discrete&#160;intraneuronal fine-particulate staining (more evident in the hypoglossal nucleus and accessory cuneate nucleu...

Watch this Scientific Journal Video about Detection of Abnormal Prion Protein by Immunohistochemistry at JoVE.com

Detection of Abnormal Prion Protein by Immunohistochemistry

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform ...

The technique allows the confirmatory diagnosis of prion diseases by the technique PrP scrapie in tissues, analyzing the PrP scrapie types and distribution to evaluate punitive deviation to the known PrP scrapie deposition profile. The technique is standardized and uses specific antibodies to PrP scrapie, enabling the visualization of the PrP scrapie deposition in tissues. The technique also gives information about the neuroanatomical area and type of cells.Begin by placing the slides with the tissue sections in a stainless-steel staining basket. Emerge the basket in a xylene bath. After three minutes, remove and immerse the basket once again.To remove xylene and rehydrate the sections, immerse the basket in an absolute ethanol bath. After three minutes, remove and immerse the basket once again. Air dry the sections before placing them in a 90%ethanol bath for one minute.Next, transfer the section to a 70%ethanol bath for another minute. For each ethanol bath treatment, gently agitate the slide basket twice and drain the basket prior to the next transfer. Transfer the basket to a 50%ethanol bath for one minute.Carefully immerse the tissue sections in 98%formic acid at room temperature for 30 minutes. Rinse the sections in tap water for five minutes followed by rinsing twice in distilled water. Use a stainless-steel staining container in the pressure chamber filled with 500 milliliters of distilled water to pretreat 10-millimolar citrate buffer at 98 degrees Celsius for 20 minutes.For quality control purposes, place a section of adhesive autoclave indicator tape on the basket to monitor the temperature and pressure. Once the alarm indicates that the equipment has attained the program time and temperature, immerse the basket with slides. Next, initiate the program on the pressure chamber to Set Point 2 and record the initial and final pressure of this program.For the inactivation of endogenous peroxidase, immerse the slide basket containing the sample sections in a bath of 3%hydrogen peroxide in methanol for 30 minutes. Bridge the sections under running water for five minutes After draining, immerse the sections in Tris-buffered saline for an additional five minutes. For immunodetection, place each slide onto a commercially available cover-plate holder pre-moistened with Tris-buffered saline with the tissue side facing the holder and slide edges coinciding with the two lower points of the holder.Ensure to avoid air bubbles. Hold the slide-holder assembly between the thumb and forefinger. Keep one finger on top of the sample slide and the other on the bottom of the holder, then place the assembly in the gallery of the system.To ensure that the set is well assembled, fill the well between the sample slide and the holder with Tris-buffered saline without overflowing it. From this point, ensure that approximately 80 microliters of Tris-buffered saline must be retained between the holder and the slide. Do not allow the sections to dry.Decrease the background staining prior to the treatment with the primary antibody by pre-incubating the slide samples for 30 minutes with 20%normal serum from the same species as the secondary antibody host in Tris-buffered saline. Without washing the sections, apply 200 microliters of the primary antibody solution directly into each well of the slide-holder set and incubate for 60 minutes at room temperature. To wash the sections, fill the wells with Tris-buffered saline, and wait for five minutes.Repeat the wash twice. Next, dilute the biotinylated secondary antibody at 1-to-200 concentration in Tris-buffered saline with 10%horse serum. Repair the required volume depending on the number of sections to be treated.Apply 200 microliters of secondary antibody solution to each well of the slide-holder set. After 30 minutes of incubation at room temperature, repeat the Tris-buffered saline wash. For incubation with avidin-biotin complex peroxidase, prepare the reagent 30 minutes before use and apply 200 microliters of the solution in each well of the slide-plate holder.Once done, incubate the plate-holder set for 30 minutes at room temperature. The level of non-specific immunolabeling in known transmissible spongiform encephalopathies-negative control animals was determined to interpret the specific abnormal prion proteins immunolabeling properly. Non-target labeling by anti-PrP antibodies was noted to occur as discrete intraneuronal fine-particulate staining, irregular fine linear threads of stain in the neuropil, diffuse non-particulate labeling in some gray and white matter areas, and diffuse cytoplasmic labeling of neurons.The table summarizes the description of immunolabeled abnormal prion protein types observed in bovine spongiform encephalopathy, classical and atypical scrapie, and a chronic wasting disease of cervids for a positive diagnosis and established criteria for negative, inconclusive, and unsuitable results. All the steps are very important. The pH of the solutions and the room temperature are important features for the success of this technique.Developing immunohistochemistry to detect both PrP scrapie and type of cells can give information about each cell involved in prime pathogenesis.

Research

JoVE Journal

Neuroscience

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...