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

この記事は、FCT(Fundação para a Ciência e a Tecnologia)-FEDER -Balcão2020の支援を受けたプロジェクトPOCI-01-0145-FEDER-029947「ポルトガルにおける慢性消耗性疾患リスク評価」によって資金提供されました。また、研究ユニットCECAVの著者は、プロジェクトUIDB/CVT/0772/2020の下でFCTから資金提供を受けました。

ここに記載されているIHC手順は、研究プロジェクトFAIRJ-CT98-7021「反芻動物TSEの監視のためのヨーロッパネットワークの確立、および疑わしい症例を特定するためのプロセスと基準の標準化と調和」の構成要素です。これは、世界獣疫事務局、WOAH(以前は国際版動物事務局、OIE)1および動物TSEの欧州参照研究所8,11によって定義された...

<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>

TSEは潜在的な人獣共通感染症です。1986年に英国でBSEが出現した後、ポルトガルはこの病気の発生率が高い欧州連合加盟国の1つになりました14,15。この病気を制御するために、出現した他のTSE(古典的および非定型のスクレイピー、BSEバリアント、および現在は子宮頸管の慢性消耗性疾患のサーベイランス)、食品獣医総局(DGAV)および国立農業獣医研?...

プリオン仮説によれば、異常なアイソフォーム(PrPSc)は、伝染性海綿状脳症(TSE)における感染因子の主要な、または唯一の成分です。TSEの診断の確認は、脳組織の免疫組織化学(IHC)プロトコルおよび/またはウェスタンイムノブロット法(WB)の適用によるPrPSc の免疫検出に依存しています1。
IHCは、組織切片の細胞にある目的の特異的標的抗原の免疫染色の最初のステップとして、モノクローナル抗体または場合によってはポリクローナル抗体(一次抗体として)を使用する方法です。次に、一次抗体に特異的な二次抗体を使用して、有効な一次抗体-抗原結合を視覚化します。これらの二次抗体は、西洋ワサビペルオキシダーゼ(HRP)やアルカリホスファターゼ(AP)などの酵素に結合しています。次に、これらの酵素に基質を添加し、一次抗体が標的抗原に結合している領域に局在する不溶性カラー生成物を生成することによって、視覚化が達成されます。改善された可視化は、対比染色によって達成することができ、ここで、色素は、免疫標識組織と非免疫標識組織との間のコントラストを作り出すために使用される2。
ホルマリン固定パラフィン包埋組織(FFPE)を用いたIHCでは、ホルマリン固定は、ホルムアルデヒドによる架橋やパラフィン包埋時の加熱脱水により、一次抗体の有効性を無効にする可能性があります。これらはタンパク質の立体構造を変化させ、エピトープを破壊、変性、またはマスクし、したがってそれらの検出を減少または消失させる3。そのため、これには抗原賦活化(AR)が必要です。AR技術は、抗原分子内のホルムアルデヒド関連の化学基架橋を破壊し、元の抗原-タンパク質立体構造を復元またはマスク解除します。これにより、免疫標識に対する抗体抗原(エピトープ)親和性が回復します。ARの最終的な有効性は、標的抗原および/または一次抗体の品質に依存します2。
熱誘導抗原(エピトープ)賦活化(HIER)は、AR3 の1つの手順であり、本明細書に記載されるように、PrPSc IHC検出のために日常的に使用される。IHCは診断に不可欠であり、病理学関連抗原の組織分布を決定するために研究室で使用されます。がん、神経科学、感染症の診断と研究に広く使用されています4。東証にとってIHCは、自然宿主や実験モデルにおけるPrPSc 分布を確認・調査するための診断・研究において重要な役割を果たしています。IHCは、プリオン病因研究およびPrPSc 沈着のタイプおよびパターンの分析、すなわち神経組織5において、日常的に記載されている感染からの逸脱を検出し、推定される新しいプリオン株を特定することに貢献する。
牛海綿状脳症(BSE)のプリオンはヒトに感染する可能性があるため、BSEの作業に関与する特定の検査プロトコルでは、BSL-3施設および実践の使用が必要になる場合があります6。これには、BSE感染の可能性のある組織サンプルを研究所や実験室内で輸送するための密閉された二次容器の使用が含まれます。また、可能な限り、BSEの研究と分析のための封じ込めエリアとプリオン専用機器を指定することも含まれます。これは、作業エリア外の汚染を防ぎ、除染手順が必要になるため、限られたスペースを提供するために行われます。
したがって、INIAVの病理学研究所は、TSEサーベイランスに関連する牛、小型反芻動物、および子宮頸管からの組織の潜在的なプリオン感染サンプルを管理するために、推奨されるバイオセーフティレベル3(BSL-3)施設および慣行6 に従います。
TSEの診断または研究手順に含まれるホルマリン固定およびパラフィン包埋組織は、特に中枢神経系において、潜在的に感染性である可能性があります。したがって、これらの固定組織は、組織処理の前にプリオンが存在する場合は、その感染力を低下させるためにギ酸で処理する必要があります。これは、固定されたトリミングされた組織(厚さ約2〜4 mm)を加工カセットに入れることによって行われます。次いで、カセットを98%ギ酸に浸す(1時間)。浸漬後、ティッシュを含むカセットを水道水で30分間洗浄し、さらに処理する前に固定液に戻します。組織切片が処理前に処理されない場合、組織学的染色の前に、ミクロトミック後の組織切片を希釈されていないギ酸に少なくとも5分間浸す必要があります7。PrPSc のためのIHCプロトコルは、プリオン7を不活性化する役割を果たすルーチンのギ酸エピトープデマスキング工程を含む。これらのプリオン不活性化ステップの後、得られた固定組織は、次いで、標準的なBSL-2慣行を用いてBSL-2で処理することができる。
TSEサーベイランスに含まれる動物のTSE診断の最小組織サンプリング要件は、脳幹を(obexレベルで)収集することです。さらに、非定型BSEとスクレイピーを検出するには、小脳の一部も収集することをお勧めします1,8。CWD診断では、脳幹(obex)と後咽頭リンパ節の両方を検査する必要があります PrP Scはリンパ組織で検出され、obex9では検出可能なPrPScがなかったため、Machado et al.10によってレビューされました。
脳幹の肛骨部分は、診断用TSE標的部位、すなわち、背迷走神経核(DVN)、孤立性路核(STN)および三叉神経の脊髄核(V)を含む。これらの地域は、BSEや古典的なスクレイピーの初期段階においても、一貫して二国間のPrPSc 蓄積を示しています。進行性TSEの臨床例では、脳幹内のすべての灰白質領域が広範囲のPrPSc 分布を示しています11。
切片作成と処理の前に、脳サンプルを評価し(図1)、自己消化のレベルと、IHCベースの確認診断へのサンプルの適合性を損なう可能性のある組織損傷の存在を確認します8。分取プロトコルと分析結果の完全性を検証するために、TSE陽性および陰性組織サンプルは、各アッセイのテストケースからの組織の調製と併せてコントロールとして含まれます。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64560/64560fig01.jpg"> 図1:PrPSc IHC手順。 組織切片の脱パラフィンから最終的な免疫染色および検出までのPrPSc IHC手順の段階的なシーケンスを示す表現(FFPE-ホルマリン固定パラフィン包埋;マブ - モノクローナル抗体。DAB - 3,3'ジアミノベンジジン)。この図は BioRender.com 年に作成されました。 <a href="https://www.jove.com/files/ftp_upload/64560/64560fig01large.jpg" target="_blank">この図の拡大版を表示するには、ここをクリックしてください。</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

異常プリオンタンパク質(PrPSc)は、細胞プリオンタンパク質の疾患関連アイソフォームであり、伝染性海綿状脳症(TSE)の診断マーカーです。これらの神経変性疾患は、ヒトおよびいくつかの動物種に影響を及ぼし、スクレイピー、人獣共通感染症のウシ海綿状脳症(BSE)、子宮頸部の慢性消耗性疾患(CWD)、および新たに特定されたラクダプリオン病(CPD)が含まれます。TSEの診断は、脳組織、すなわち脳幹(obexレベル)に対する免疫組織化学(IHC)およびウェスタンイムノブロット法(WB)の両方の適用によるPrPSc の免疫検出に依存しています。IHCは、組織切片の細胞における目的の抗原に対する一次抗体(モノクローナルまたはポリクローナル)を使用する広く使用されている方法です。抗体-抗原結合は、抗体が標的となった組織または細胞の領域に局在したままの呈色反応によって視覚化することができる。このように、プリオン病では、他の研究分野と同様に、免疫組織化学技術は診断目的だけでなく、病因研究にも使用されます。このような研究は、新しいプリオン株を同定するために、前述のものからPrPSc パターンおよびタイプを検出することを含む。BSEはヒトに感染する可能性があるため、東証サーベイランスに含まれる牛、小型反芻動物、および子宮頸管サンプルの取り扱いには、バイオセーフティ研究所レベル3(BSL-3)の施設および/または診療所を使用することが推奨されます。さらに、汚染を制限するために、可能な限り封じ込めおよびプリオン専用装置が推奨されます。PrPSc IHC手順は、この技術で使用されるホルマリン固定およびパラフィン包埋組織が感染性のままであるため、プリオン不活化手段としても機能するギ酸エピトープデマスキングステップで構成されています。結果を解釈する際には、非特異的免疫標識と標的標識を区別するように注意する必要があります。この目的のために、既知のTSE陰性対照動物において得られた免疫標識のアーティファクトを認識し、それらを特定のPrPSc 免疫標識タイプから区別することが重要であり、これはTSE株、宿主種、および prnp 遺伝子型の間で異なり得る。

非特異的標的免疫標識が可能であることを考えると、既知のTSE陰性対照動物における非特異的免疫標識のレベルを確認することが重要です。これは、特異的なPrPSc 免疫標識7 (図2)を適切に解釈するための重要なステップである。 抗PrP抗体による非標的標識は、離散的な神経内微粒子染色(舌下核および副核でより顕著)、ニューロパイルの...

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

Detection of Abnormal Prion Protein by Immunohistochemistry

免疫組織化学プロトコルを使用して異常なプリオンタンパク質を免疫標識するには、特定のサンプルおよび抗PrP抗体調製方法が必要です。本プロトコルは、適切なPrP免疫標識を確保し、非特異的バックグラウンド染色を最小限に抑えるためのエピトープデマスキングの重要なステップを説明しています。また、このアプローチは、プリオン感染組織を用いて免疫組織化学研究を行う際のバイオセーフティ対策を考慮する。

免疫組織化学による異常プリオンタンパク質の検出

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

この技術は、組織中のPrPスクレイピーの技術によるプリオン病の確認診断を可能にし、既知のPrPスクレイピー沈着プロファイルに対する懲罰的偏差を評価するためにPrPスクレイピーの種類および分布を分析する。この技術は標準化されており、PrPスクレイピーに対する特異的抗体を使用しており、組織におけるPrPスクレイピー沈着の可視化を可能にします。この技術はまた、神経解剖学的領域および細胞の種類に関する情報を提供する。まず、組織切片を含むスライドをステンレス鋼の染色バスケットに入れます。バスケットをキシレン浴に入れます。3分後、バスケットを取り外してもう一度浸します。キシレンを除去して切片を再水和するには、バスケットを絶対エタノール浴に浸します。3分後、バスケットを取り外してもう一度浸します。切片を風乾してから、90%エタノール浴に1分間入れます。次に、切片を70%エタノール浴にさらに1分間移します。エタノール浴処理ごとに、スライドバスケットを2回静かに攪拌し、次の移送の前にバスケットを排出します。バスケットを50%エタノールバスに1分間移します。組織切片を室温で98%ギ酸に30分間注意深く浸します。切片を水道水で5分間すすぎ、蒸留水で2回すすぎます。500ミリリットルの蒸留水で満たされた圧力チャンバー内のステンレス鋼染色容器を使用して、10ミリモルのクエン酸塩バッファーを摂氏98度で20分間前処理します。品質管理の目的で、バスケットに粘着性オートクレーブインジケータテープのセクションを置き、温度と圧力を監視します。機器がプログラムの時間と温度に達したことをアラームが示したら、バスケットをスライドに浸します。次に、圧力室でプログラムを開始してセットポイント2にし、このプログラムの初期圧力と最終圧力を記録します。内因性ペルオキシダーゼを不活性化するには、サンプル切片を含むスライドバスケットをメタノール中の3%過酸化水素浴に30分間浸します。切片を流水で5分間橋渡しします 排水後、切片をトリス緩衝生理食塩水にさらに5分間浸します。免疫検出のために、各スライドをトリス緩衝生理食塩水であらかじめ湿らせた市販のカバープレートホルダーに置き、組織側をホルダーに向け、スライドの端をホルダーの2つの下部ポイントと一致します。気泡を避けてください。スライドホルダーアセンブリを親指と人差し指で挟みます。片方の指をサンプルスライドの上に置き、もう片方の指をホルダーの下部に置き、アセンブリをシステムのギャラリーに置きます。セットが適切に組み立てられていることを確認するために、サンプルスライドとホルダーの間のウェルに、トリス緩衝生理食塩水をオーバーフローせずに充填します。この時点から、約80マイクロリットルのトリス緩衝生理食塩水をホルダーとスライドの間に保持する必要があることを確認してください。切片を乾かさないでください。トリス緩衝生理食塩水中で二次抗体宿主と同じ種の20%正常血清でスライドサンプルを30分間プレインキュベートすることにより、一次抗体で処理する前にバックグラウンド染色を減らします。切片を洗浄せずに、200マイクロリットルの一次抗体溶液をスライドホルダーセットの各ウェルに直接塗布し、室温で60分間インキュベートします。切片を洗浄するには、ウェルにトリス緩衝生理食塩水を満たし、5分間待ちます。洗浄を2回繰り返します。次に、ビオチン化二次抗体をトリス緩衝生理食塩水中で1〜200濃度で10%ウマ血清で希釈します。治療するセクションの数に応じて必要な量を修復します。200マイクロリットルの二次抗体溶液をスライドホルダーセットの各ウェルに塗布します。室温で30分間インキュベートした後、トリス緩衝生理食塩水洗浄を繰り返す。アビジン-ビオチン複合体ペルオキシダーゼとのインキュベーションのために、使用の30分前に試薬を調製し、スライドプレートホルダーの各ウェルに200マイクロリットルの溶液を塗布する。完了したら、プレートホルダーセットを室温で30分間インキュベートします。既知の伝染性海綿状脳症陰性対照動物における非特異的免疫標識のレベルは、特異的異常プリオンタンパク質免疫標識を適切に解釈するために決定された。抗PrP抗体による非標的標識は、離散的なニューロン内微粒子染色、ニューロパイルの不規則な微細な線状染色糸、一部の灰白質および白質領域におけるびまん性非粒子標識、およびニューロンのびまん性細胞質標識として発生することが指摘されました。この表は、ウシ海綿状脳症、古典的および非定型のスクレイピー、および陽性診断のための子宮頸部の慢性消耗性疾患で観察される免疫標識異常プリオンタンパク質タイプの説明と、陰性、不確定、および不適切な結果の確立された基準を要約したものです。すべてのステップは非常に重要です。溶液のpHと室温は、この技術の成功にとって重要な特徴です。PrPスクレイピーと細胞の種類の両方を検出する免疫組織化学を開発することで、主要な病因に関与する各細胞に関する情報を得ることができます。

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

Research

JoVE Journal

Neuroscience

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 two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

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

A combination of different techniques to maximize data collection from mouse tissue is presented.

親油性色素を組み合わせることで、その場でハイブリダイゼーション、免疫組織化学、および組織学

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

神経科学

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. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

内在性NADH蛍光の二光子イメージングによるマウス大脳皮質におけるMicroregional低酸素の検出

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 relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

複雑な脳の地形を明らかにするためWholemount免疫組織化学

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

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

幼虫、蛹および成人の解剖と免疫組織化学<em&gt;ショウジョウバエ</em&gt;網膜

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)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

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

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

免疫沈降およびアシル-ビオチン所（阿部）による培養海馬神経細胞におけるタンパク質パルミトイル化の検出

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

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

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

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

の検出<em&gt;その場で</emで&gt;タンパク質 - タンパク質複合体<em&gt;ショウジョウバエ</em近接ライゲーションアッセイの使用&gt;幼虫神経筋接合部

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 prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

α-シヌクレイン線維の末梢接種後のトランスジェニックマウスにおける神経炎症の生物発光イメージング

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

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

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

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

キイロショウジョウバエにおけるプリオン様蛋白質集合体の細胞間伝達を監視

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 the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

免疫組織化学準備 ImageJ を用いた組織の顕微鏡写真からミクログリアの形態の定量化

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 is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

生体内における脳タンパク質合成の局所速度の決定のための定量的自動放射線法

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