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

本文由FCT（科学和技术基金会）支持的POCI-01-0145-FEDER-029947项目“葡萄牙的慢性消耗性疾病风险评估”资助 - FEDER-Balcão2020。此外，研究单位CECAV的作者在UIDB / CVT / 0772 / 2020项目下获得了FCT的资助.我们感谢美国农业部西部区域研究中心研究主任（退休）Bruce C. Campbell的帮助。

本文所述的IHC程序是研究项目FAIRJ-CT98-7021的组成部分，“建立欧洲反刍动物TSE监测网络以及识别疑似病例的程序和标准的标准化和协调”。它遵循世界动物卫生组织、WOAH（以前称为国际动物卫生组织办公室，OIE）1 和欧洲动物 TSE8，11 参考实验室定义的诊断标准。自2008年以来，所述的IHC程序也根据NPISOIEC17025（测试和校准实验室能力的?...

<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. 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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>

TSE是潜在的人畜共患疾病。1986年英国出现疯牛病后，葡萄牙成为该病发病率较高的欧盟成员国之一14，15。为了控制这种疾病，已经出现的其他TSE（经典和非典型搔痒症，疯牛病变体，以及目前对宫颈慢性消耗性疾病的监测），食品和兽医总局（DGAV）和国家农业和兽医研究所（IP）开发了监测机制，该研究所作为TSE国家兽医实验室进行TSE诊断。该实验室...

根据朊病毒假说，异常亚型（PrPSc）是传染性海绵状脑病（TSE）中感染因子的主要或唯一成分。TSE 的诊断确认依赖于通过应用脑组织的免疫组织化学 （IHC） 方案和/或蛋白质免疫印迹法 （WB） 对 PrPSc 进行免疫检测1。
IHC是一种采用单克隆抗体或在某些情况下使用多克隆抗体（作为一抗）作为对位于组织切片细胞中的特定目标抗原进行免疫染色的第一步的方法。然后使用针对一抗的二抗观察任何有效的一抗-抗原结合。这些二抗与辣根过氧化物酶（HRP）或碱性磷酸酶（AP））偶联。然后通过向这些酶添加底物来实现可视化，产生位于一抗与靶抗原结合的区域的不溶性颜色产物。改进的可视化可以通过复染来实现，其中染料用于在免疫标记和非免疫标记的组织之间产生对比2。
对于使用福尔马林固定石蜡包埋组织 （FFPE） 的 IHC，福尔马林固定可能会由于甲醛交联以及石蜡包埋过程中的加热和脱水而使一抗的有效性失效。这些改变蛋白质的构象，破坏、变性或掩盖表位，从而减少或取消其检测3。因此，这需要抗原修复（AR）。AR技术破坏抗原分子中与甲醛相关的化学基团交联，从而恢复或揭示原始的抗原 - 蛋白质构象。这导致恢复抗体-抗原（表位）亲和力以进行免疫标记。AR的最终疗效取决于靶向抗原和/或一抗的质量2。
热诱导抗原（表位）修复（HIER）是AR3 的一个程序，常规用于PrPScIHC 检测，如本文所述。IHC 对于诊断至关重要，在研究实验室中用于确定病理相关抗原的组织分布。它广泛用于诊断和研究癌症、神经科学和传染病4 等。对于TSEs，IHC在诊断和研究中起着重要作用，以确认和研究PrPSc 在自然宿主和实验模型中的分布。IHC有助于朊病毒发病机制研究和PrPSc 沉积类型和模式的分析，即在神经组织中5中，以检测与常规描述的感染的偏差并识别推定的新朊病毒株。
由于牛海绵状脑病（BSE）的朊病毒可以感染人类，因此涉及BSE工作的某些实验室方案可能需要使用BSL-3设施和实践6。这些措施包括使用密封的二级容器在研究所和实验室内运输潜在的BSE感染组织样本。它还包括尽可能为疯牛病研究和分析指定遏制区域和朊病毒专用设备。这样做是为了防止工作区域以外的污染，并提供一个密闭的空间，因为净化程序变得必要。
因此，INIAV 病理学实验室遵循推荐的生物安全 3 级 （BSL-3） 设施和实践6 ，以管理与 TSE 监测相关的牛、小反刍动物和子宫颈的潜在朊病毒感染组织样本。
TSE诊断或研究程序中包含的福尔马林固定和石蜡包埋组织，特别是在中枢神经系统中，可能具有潜在的传染性。因此，在组织处理之前，必须用甲酸处理这些固定组织，以降低朊病毒（如果存在）的感染性。这是通过将固定的，修剪的组织（约2-4毫米厚）放置在处理盒中来完成的。然后将盒浸入98%甲酸中（1小时）。浸泡后，将带有组织的盒在流动的自来水中洗涤30分钟，并在进一步处理之前返回固定剂。如果在处理前未处理组织切片，则在组织学染色之前，必须将切片后的切片浸入未稀释的甲酸中至少5分钟7。PrPSc 的IHC方案包括常规的甲酸表位去掩蔽步骤，也用于灭活朊病毒7。在这些朊病毒灭活步骤之后，可以使用标准的BSL-2实践在BSL-2下处理所得的固定组织。
TSE监测中包含的任何动物TSE诊断的最低组织采样要求是收集脑干（在obex水平）。此外，为了检测非典型疯牛病和搔痒症，建议还应收集部分小脑1，8。对于CWD诊断，应检测脑干（obex）和咽后淋巴结，因为PrPSc可以在淋巴组织中检测到，而在obex9中没有检测到PrPSc，由Machado等人审查10。
脑干的obex部分包括诊断性TSE靶位点，即迷走神经背侧核（DVN）、孤立束核（STN）和三叉神经的脊髓束核（V）。这些区域始终呈现双侧PrPSc 积累，即使在疯牛病和经典搔痒症的早期阶段也是如此。在晚期 TSE 的临床病例中，脑干内的所有灰质区域都显示出广泛的 PrPSc 分布11。
在切片和处理之前，对脑样本进行评估（图1），以确定自溶水平和是否存在任何可能损害样本适用于基于IHC的确认性诊断的组织损伤8。为了验证制备方案和分析结果的完整性，将TSE阳性和阴性组织样品作为对照包括在内，并在每次测定中从测试用例制备组织。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg"> 图1：PrPSc免疫 接种程序。 表示显示从组织切片脱蜡到最终免疫染色和检测的 PrPSc IHC 程序的分步顺序（FFPE - 福尔马林固定石蜡包埋;Mab - 单克隆抗体;DAB - 3，3' 二氨基联苯胺）。这个数字是在 BioRender.com 年创建的。 <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">请点击此处查看此图的大图。</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

异常朊蛋白 （PrPSc） 是细胞朊蛋白的疾病相关亚型，也是传染性海绵状脑病 （TSE） 的诊断标志物。这些神经退行性疾病影响人类和几种动物物种，包括搔痒症、人畜共患牛海绵状脑病 （BSE）、宫颈慢性消耗性疾病 （CWD） 和新发现的骆驼朊病毒病 （CPD）。TSE的诊断依赖于通过对脑组织（即脑干）应用免疫组织化学（IHC）和西方免疫印迹法（WB）对PrPSc 进行免疫检测。IHC是一种广泛使用的方法，它使用一抗（单克隆或多克隆）来对抗组织切片细胞中的目标抗原。抗体-抗原结合可以通过颜色反应来可视化，该颜色反应仍局限于抗体靶向的组织或细胞区域。因此，在朊病毒疾病中，与其他研究领域一样，免疫组织化学技术不仅用于诊断目的，还用于发病机制研究。此类研究涉及从先前描述的模式和类型中检测PrPSc 模式和类型，以鉴定新的朊病毒菌株。由于疯牛病可以感染人类，建议使用生物安全实验室3级（BSL-3）设施和/或实践来处理TSE监测中包含的牛，小反刍动物和宫颈样本。此外，建议尽可能使用密闭和朊病毒专用设备，以限制污染。PrPSc IHC程序包括甲酸表位去掩蔽步骤，也作为朊病毒灭活措施，因为该技术中使用的福尔马林固定和石蜡包埋组织仍然具有传染性。在解释结果时，必须注意区分非特异性免疫标记和靶标标记。为此，重要的是要识别在已知的TSE阴性对照动物中获得的免疫标记伪影，以区分这些伪影与特定的PrPSc 免疫标记类型，其可以在TSE菌株，宿主物种和 prnp 基因型之间变化，本文进一步描述。

鉴于非特异性靶标免疫标记是可能的，因此确定已知TSE阴性对照动物的非特异性免疫标记水平非常重要。这是正确解释特异性PrPSc免疫标记7的重要步骤（图2）。 抗 PrP 抗体的非靶标标记表现为离散的神经元内细颗粒染色（在舌下核和辅助楔形核中更明显）、神经枕中不规则的细线染色（STN、V、寡核、附属楔形核、侧束核、寡核背侧网状形成...

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

Detection of Abnormal Prion Protein by Immunohistochemistry

使用免疫组织化学方案对异常朊蛋白进行免疫标记需要特定的样品和抗PrP抗体制备方法。本协议描述了表位去掩膜的关键步骤，以确保适当的PrP免疫标记并最大限度地减少非特异性背景染色。此外，这种方法在对朊病毒感染的组织进行免疫组织化学研究时考虑了生物安全措施。

免疫组化检测异常朊蛋白

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

该技术允许通过组织中的PrP搔痒症技术对朊病毒病进行确认性诊断，分析PrP搔痒症的类型和分布，以评估与已知PrP搔痒症沉积曲线的惩罚性偏差。该技术是标准化的，并使用针对PrP搔痒症的特异性抗体，使PrP搔痒症在组织中的沉积可视化。该技术还提供有关神经解剖区域和细胞类型的信息。首先将带有组织切片的载玻片放入不锈钢染色篮中。将篮子放入二甲苯浴中。三分钟后，取出篮子并再次浸入。要去除二甲苯并使切片再水化，请将篮子浸入无水乙醇浴中。三分钟后，取出篮子并再次浸入。风干切片，然后将其放入90%乙醇浴中一分钟。接下来，将该部分转移到70%乙醇浴中再发酵一分钟。对于每次乙醇浴处理，轻轻搅拌载玻片篮两次，并在下一次转移之前排干篮子。将篮子转移到50%乙醇浴中一分钟。在室温下小心地将组织切片浸入98%甲酸中30分钟。用自来水冲洗切片五分钟，然后在蒸馏水中冲洗两次。在装有500毫升蒸馏水的压力室中使用不锈钢染色容器，在98摄氏度下预处理10毫摩尔柠檬酸盐缓冲液20分钟。出于质量控制目的，在篮子上放置一段粘性高压釜指示胶带以监测温度和压力。一旦警报表明设备已达到编程时间和温度，请用载玻片浸入篮子。接下来，在压力室上启动程序以设定点 2 并记录该程序的初始和最终压力。对于内源性过氧化物酶的灭活，将装有样品部分的载玻片篮浸入3%过氧化氢甲醇浴中30分钟。在流水下桥接部分五分钟 排水后，将部分浸入Tris缓冲盐水中再浸泡五分钟。对于免疫检测，将每张载玻片放在市售的盖板支架上，该支架预先用Tris缓冲盐水润湿，组织侧朝向支架，载玻片边缘与支架的两个下点重合。确保避免气泡。将滑动支架组件夹在拇指和食指之间。将一根手指放在样品载玻片的顶部，另一根手指放在支架的底部，然后将组件放在系统的库中。为确保套件组装良好，请用Tris缓冲盐水填充样品载玻片和支架之间的孔，不要溢出。从这一点开始，确保在支架和载玻片之间必须保留大约 80 微升的 Tris 缓冲盐水。不要让切片干燥。通过在Tris缓冲盐水中将载玻片样品与二抗宿主相同物种的20%正常血清预孵育30分钟，减少一抗处理前的背景染色。在不清洗切片的情况下，将200微升一抗溶液直接涂入载玻片支架组的每个孔中，并在室温下孵育60分钟。要清洗切片，请用Tris缓冲盐水填充孔，然后等待五分钟。重复洗涤两次。接下来，用 10% 马血清在 Tris 缓冲盐水中以 1 至 200 浓度稀释生物素化的二抗。根据要处理的部分数量修复所需的体积。将200微升二抗溶液应用于载玻片支架组的每个孔中。在室温下孵育30分钟后，重复Tris缓冲盐水洗涤。为了与亲和素 - 生物素复合物过氧化物酶孵育，在使用前30分钟准备试剂，并在滑板支架的每个孔中施用200微升溶液。完成后，将板架在室温下孵育30分钟。测定已知传染性海绵状脑病阴性对照动物的非特异性免疫标记水平，以正确解释特异性异常朊蛋白免疫标记。注意到抗PrP抗体的非靶标标记表现为离散的神经元内细颗粒染色，神经枕中不规则的细线染色，某些灰质和白质区域的弥漫性非颗粒标记以及神经元的弥漫性细胞质标记。该表总结了在牛海绵状脑病、经典和非典型搔痒症以及宫颈慢性消耗性疾病中观察到的免疫标记异常朊蛋白类型的描述，以做出阳性诊断，并建立了阴性、不确定和不合适结果的标准。所有步骤都非常重要。溶液的pH值和室温是该技术成功的重要特征。开发免疫组织化学来检测PrP搔痒症和细胞类型可以提供有关参与主要发病机制的每个细胞的信息。

Research

JoVE Journal

Neuroscience

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

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

结合亲脂性染料，原位杂交，免疫组织化学和组织学

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

科研

神经科学

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

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

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

为揭示复杂的大脑地形wholemount免疫组化

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

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

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

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（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

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

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

在转基因小鼠神经炎症的生物发光成像的α-共核蛋白纤丝的外围接种后

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

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

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

应用 ImageJ 对免疫组化制备组织显微照片小胶质细胞形态学的定量研究

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

定量自动放射法测定维沃脑蛋白合成区域速率

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