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 TSEs8,11. The IHC procedure described is also accredited according to NPISOIEC17025 (general requirements for the competence of testing and calibration laboratories) since 2008. Such accreditation implies a high-quality management system incorporating standard operative procedures, qualified personnel, accurately calibrated equipment, rigorous control of data and reagents, participation in inter-laboratory proficiency assays, non-conforming work, and risk assessment to increase the effectiveness of the management system, achieving improved results, and preventing laboratory hazards. Prion-infected tissue controls with BSE, classical scrapie, atypical scrapie, and CWD were collected (formalin-fixed, paraffin-embedded, FFPE) from various sources. The bovine and sheep sections are from the cases diagnosed under an animal TSE surveillance program. The CWD sections are from the control samples kindly provided by Professor Stefanie Czub (Canadian Food Inspection Agency, National Center for Animal Diseases) in 2003 and from the CWD Proficiency testing 2008 organized by the European Reference Laboratory for TSEs (APHA, Weybridge).
1. Tissue sectioning and slide preparation
<ol>
	<li>Cut sections of formalin-fixed, paraffin-embedded (FFPE) tissues at 3-5 &#181;m thickness using a microtome.</li>
	<li>Float sections onto purified water with a temperature approximately 10 &#730;C below the melting point of the paraffin used. Lift the sections from the water onto specially treated microscope slides (see Table of Materials). Allow the water to drain thoroughly from the slides.</li>
	<li>Incubate the slides overnight at 50 &#730;C to enhance the slide adhesion of tissues. 
	NOTE: It is preferable to prepare freshly sectioned tissues for IHC protocols, although TSE positive and negative control sections can be prepared in advance and stored. Steps 2 to 4 must be performed in a chemical fume hood.</li>
</ol>
2. Deparaffinization and rehydration
<ol>
	<li>Place the slides with the tissue sections into a stainless-steel staining basket (see Table of Materials). Immerse the basket into a xylene bath (stainless-steel staining container) for 3 min, remove and immerse once again.</li>
	<li>Remove xylene and rehydrate sections by immersing them in an absolute ethanol bath for 3 min. Remove and immerse once again.</li>
	<li>Air dry the sections and place in a 90% ethanol bath for 1 min. Transfer to a 70% ethanol bath for another minute, followed by a final transfer to a 50% ethanol bath for 1 min. For each ethanol bath treatment, gently agitate the slide basket twice and drain the basket prior to the next transfer.</li>
</ol>
3. Epitope retrieval
<ol>
	<li>Carefully immerse the tissue sections in 98% formic acid at room temperature for 30 min. Rinse the sections in tap water for 5 min, followed by rinsing twice in distilled water. 
	CAUTION: Formic acid is highly corrosive. Protective goggles and gloves must be worn.</li>
	<li>Perform Heat-Induced Antigen Retrieval (HIER) following the steps below. 
	NOTE: This step is performed by hydrated autoclaving at 121 &#176;C for 30 min in a specific pressure chamber (see Table of Materials).
	<ol>
		<li>Pre-heat the citrate buffer (10 mM, pH 6.1, see Table of Materials) at 98 &#176;C for about 20 min in a stainless-steel staining container placed inside the pressure chamber filled with 500 mL of distilled water. 
		NOTE: For the present study, the program Set Point was 1 (SP1) for the specific equipment used.</li>
		<li>After the alarm indicates that the equipment attained the programmed time and temperature, immerse the basket with slides and initiate the program to Set Point 2 (121&#176; C for 30 min). For quality control purposes, place a section of adhesive autoclave indicator tape on the basket to monitor the temperature and pressure, and record the initial and final pressure of this program.</li>
		<li>If the number of slides to be immunostained does not reach the basket&#39;s capacity, use clean, blank slides to occupy any empty positions. After the program has terminated, allow the passive return of the chamber to ambient pressure (at least 30 min). 
		NOTE: Longer durations may reduce non-specific background staining.</li>
		<li>Carefully transfer the slide basket to a stainless-steel staining container filled with distilled water for 5 min.</li>
	</ol>
	</li>
</ol>
4. Inactivation of endogenous peroxidase
<ol>
	<li>Immerse the slide basket containing the sample sections in a bath of 3% hydrogen peroxide (H2O2) in methanol for 30 min. Rinse the sections under running water (5 min). Drain and immerse the sections in 1x Tris-buffered saline (TBS) for an additional 5 min.</li>
</ol>
5. Immunodetection
NOTE: For the present study, immunodetection was executed using the capillary gap format7 using a commercially available slide clip assembly system (see Table of Materials). Other immunohistochemistry slide systems are also applicable.
<ol>
	<li>Place each slide onto a commercially available cover-plate holder (see Table of Materials) pre-moistened with TBS, avoiding bubbles, with the tissue side facing the holder and slide edges coinciding with the two lower points of the holder.
	<ol>
		<li>Hold the slide-holder assembly between the thumb and the forefinger, with 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.</li>
		<li>To ensure the set is well assembled, fill the well between the sample slide and the holder with TBS, which must not immediately overflow. From this point on, ensure that approximately 80 &#181;L of TBS must be retained between the holder and the slide. Do not allow the sections to dry.</li>
	</ol>
	</li>
	<li>Once in the system, to decrease background staining prior to the treatment with primary antibody (anti-PrP, see Table of Materials), pre-incubate the sample slides with 20% normal serum from the same species as the secondary antibody host being used for immunostaining (in the present case, horse serum) for 30 min in TBS.</li>
	<li>Thaw the number of aliquots of antibody to be used according to the animal species under analysis, the number of sample sections to be examined, and the amount of working dilution. 
	NOTE: For best results, use 10% normal serum from the same species as the source of the secondary antibody and primary antibody solutions. If possible, for best results, the host source of the normal serum and secondary antibody should be from the same species.</li>
	<li>Without washing the sections, apply the primary antibody solution directly (200 &#181;L) in each well of the slide-holder set and incubate for 60 min at room temperature.</li>
	<li>For washing, fill the wells of the slide-holder sets with TBS and wait for 5 min. Repeat twice.</li>
	<li>Dilute the biotinylated secondary antibody (monoclonal anti-murine Horse Ab, see Table of Materials) at 1/200 in TBS with 10% horse serum. Prepare the volume required depending on the number of sections to be treated.</li>
	<li>Apply the secondary antibody solution (200 &#181;L) to each well of the slide-holder set. Incubate for 30 min at room temperature.</li>
	<li>Wash as described in step 5.5.</li>
	<li>For incubation with avidin-biotin complex peroxidase (ABC/HRP complex, see Table of Materials), prepare the reagent 30 min before use. Apply the ABC/HRP complex solution (200 &#181;L) in each well of the slide-plate holder set. Incubate for 30 min at room temperature.</li>
	<li>Wash as described in step 5.5.</li>
</ol>
6. Development with DAB chromogen
CAUTION: DAB is a potential carcinogen. As a result, appropriate care is necessary while working with this reagent, including eye protection, lab coats, gloves, and good laboratory procedures. Dispose of following local regulations.
<ol>
	<li>Dilute chromogen according to the manufacturer&#39;s instructions (see Table of Materials) immediately before use. Apply the chromogen solution (400 &#181;L) to each well of the slide-plate set.</li>
	<li>Incubate for up to 30 min at room temperature. In PrPSc positive sections, a 10 min incubation period is usually sufficient.</li>
	<li>Remove the residual chromogen solution by washing the slides in distilled water. Remove the slides from the coverplate holders and place them in a plastic container with distilled water.</li>
</ol>
7. Hematoxylin counterstaining
<ol>
	<li>Immerse the stainless-steel staining basket with the sections into the hematoxylin solution bath (see Table of Materials) for 1 min. Rinse gently in cold running water for 10 min or tepid water for 5 min.</li>
	<li>Dehydrate the sections by placing them in 90% ethanol for 1 min, followed by one bath in absolute ethanol for 1 min. Then, place the slides in a xylene bath for 1 min.</li>
	<li>Mount the sections with a commercially available mounting medium (see Table of Materials). 
	NOTE: These steps are carried out in a chemical fume hood at room temperature (19 &#730;C-24 &#730;C). This procedure includes a test record (Supplementary File 1) for recording the completion of all the steps, the equipment used, and the lab technician. In addition, a worksheet (Supplementary File 2) has been designed to easily determine the volumes of the reagents according to the number of sections in each assay, the working dilutions, and the manufacturer&#39;s instructions as well as to record the room temperature, equipment, and reagent batches.</li>
</ol>

<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>

The authors have nothing to disclose.

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 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="Immunohistochemistry workflow diagram: FFPE tissue processing, antigen retrieval, antibody staining." class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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' 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><tbody><tr><td>Absolute ethanol&amp;nbsp;</td><td>Labchem</td><td>LB0507-9010</td><td>Undituled</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>Diluted 90%, 70% and 50% in distilled water</td></tr><tr><td>Avidin-biotin complex and peroxidase&lt;br/&gt; 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)&amp;nbsp;</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&amp;nbsp;</td><td>Vector Laboratories</td><td>SK-4100</td><td>Prepare before use according to kit instructions.&lt;br/&gt; Use 400 &amp;micro;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&amp;rsquo;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&amp;nbsp;</td><td>Labchem</td><td>LB0507-9010</td><td></td></tr><tr><td>Glacial acetic acid&amp;nbsp;</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 H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;):</td><td></td><td></td><td>40 mL Hydrogen peroxide (30% w/w) in 360 mL Methanol.&lt;br/&gt; 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&amp;nbsp;</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&amp;nbsp;</td><td>Shandon-AS325</td><td>Microtome&amp;nbsp;</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:&lt;br/&gt; Horse normal serum</td><td>&lt;br/&gt; Gibco</td><td>&lt;br/&gt; 16050-122&amp;nbsp;</td><td>Prepare final volume according to the number of sections in the assay&lt;br/&gt; (200 &amp;micro;L of solution per section).</td></tr><tr><td>Primary antibody anti-PrP Mouse MAb 2G11&amp;nbsp;</td><td>BIORAD</td><td>MCA2460</td><td>PrP 146-R154R171182&lt;br/&gt; Ovine including atypical scrapie, cervine, feline. Not suitable for bovine.&lt;br/&gt; According to the number of sections in the assay (200 &amp;micro;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)&lt;br/&gt; 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 &amp;mu;L into sterile microtubes. Defrost and use one aliquot at a time.&amp;nbsp;</td></tr><tr><td>Primary antibody anti-PrP Mouse MAb 12F10&amp;nbsp;</td><td>Cayman&amp;nbsp; Chemical Company&amp;nbsp;</td><td>189710</td><td>PrP142-160&lt;br/&gt; Bovine, not suitable for ovine&lt;br/&gt; Usual antibody dilution: 1/200 but working dilution should also be established. Prepare as MAb 2G11</td></tr><tr><td>Shandon Coverplate&lt;sup&gt;TM&lt;/sup&gt; chamber</td><td>Thermo Scientific&amp;nbsp;</td><td>72110017</td><td></td></tr><tr><td>Shandon Sequenza&amp;reg; Immunstaining center&amp;nbsp;</td><td>Thermo Scientific</td><td>73300001</td><td></td></tr><tr><td>Shandon Sequenza&amp;reg; 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&amp;nbsp; and 0.255 g Citric acid in one litre purified water.&lt;br/&gt; Adjust pH of working solution to 6.1 using 10 mM citric acid solution (1.05 g citric acid in 500 mL purified water)&lt;br/&gt; Prepare on assay day.&amp;nbsp;</td></tr><tr><td>Tri-sodium citrate dihydrate</td><td>Sigma-aldrich&amp;nbsp;</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&amp;nbsp;</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):&lt;br/&gt; 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&amp;plusmn; 3 &amp;deg;C until 2 months)&lt;br/&gt; Dilute TBS stock solution 1/10 on assay day.&amp;nbsp;</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&amp;nbsp;</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 intraneuronal fine-particulate staining (more evident in the hypoglossal nucleus and accessory cuneate nucleus), ...

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 ...

detection-of-abnormal-prion-protein-by-immunohistochemistry

Research

JoVE Journal

Neuroscience

2.8K Views.  University of Trás-os-Montes and Alto Douro (UTAD). De techniek maakt de bevestigende diagnose van prionziekten mogelijk door de techniek PrP scrapie in weefsels, waarbij de PrP scrapietypes en distributie worden geanalyseerd om de bestraffende afwijking van het bekende PrP scrapie-depositieprofiel te evalueren. De techniek is gestandaardiseerd en maakt gebruik van specifieke antilichamen tegen PrP scrapie, waardoor de visualisatie van de PrP scrapie afzetting in weefsels mogelijk is. De techniek geeft ook informatie over het neuroanatomische ...

Video: Detection of Abnormal Prion Protein by Immunohistochemistry

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

Immunology and Infection

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Medicine

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Detecting Abnormal Prion Proteins in Brain Tissue Using Immunohistochemistry

Source: Orge, L., et al. <a href="https://app.jove.com/v/64560">Detection of Abnormal Prion Protein by Immunohistochemistry.</a> J. Vis. Exp. (2023).
This video demonstrates a technique to detect misfolded prion protein in brain sections using immunohistochemistry. Upon treating the brain section with formic acid to denature prions and minimize infection risk, as well as unmasking the prion aggregates using heat-induced epitope retrieval, the sections are immunolabeled for the misfolded prion protein aggregates and observed under a microscope.

1. Tissue sectioning and slide preparation

	Cut sections of formalin-fixed, paraffin-embedded (FFPE) tissues at 3-5 &#181;m thickness using a microtome.
	...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Detection of Misfolded Prion Protein Aggregates in Mouse Brain Tissue Using Western Blotting

Take tissue homogenate from a mammalian brain that contains both normal prion proteins and misfolded prion aggregates responsible for neurodegenerative diseases.
Incubate with a proteolytic enzyme to digest the normal prions, leaving the resistant aggregates intact.
Treat the aggregates with a denaturing buffer at a high temperature to linearize the proteins and impart a uniform negative charge.
Load the sample onto an electrophoresis gel and separate the proteins into bands, which confirms the presence of aggregates.
Using an electric field, transfer the separated proteins onto a membrane.
Incubate the membrane in a blocking solution to mask non-specific binding sites.
Incubate with peroxidase enzyme-tagged antibodies, which bind specifically to the prion aggregates.
Wash the membrane to remove unbound antibodies, minimizing non-specific signals.
Add a chemiluminescent substrate and an oxidizing agent, enabling the peroxidase to oxidize the substrate and produce light.
Measure the light intensity to quantify misfolded prions in the homogenate.

Detection of Abnormal Prion Protein by Immunohistochemistry

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