We thank Hans Lassmann and Jan Bauer for their guidance and support. We also thank Katalin Benedek for excellent technical assistance and Caroline Westerlund for critical and linguistic appraisal.
This study was supported by grants from Biogen Idec, the Wenner-Gren Foundation, the Swedish Research Council, the Swedish Association of Persons with Neurological Disabilities, Swedish Brain Foundation, the EU 6TH Framework EURATools (LSHG-CT-2005-019015) and Neuropromise (LSHM-CT-2005-018637). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

伦理声明 本研究是根据从瑞典国家实验动物和欧洲共同体理事会指导进行下道德许可证（86/609 / EEC）N338 / 09，N15 / 10和N65 / 10，这是由批准北斯德哥尔摩动物伦理委员会。 1.组织准备<ol><li> 灌注和固定 <ol><li>麻醉用异氟烷将动物经由左心室进行穿心灌注。发起用磷酸盐缓冲盐水（PBS）脉管系统的漂洗以除去在0.1M PBS（PFA）的血液成分，然后4％多聚甲醛。 </li><li>通过浸入PFA注视解剖大脑，脊髓和外周组织LN。之后在4℃24小时，在4℃下传输距离PFA组织到PBS和储存直至进一步处理。另外商业PBS，溶解9克氯化钠250毫升的S＆＃246;小将索伦森缓冲，并添加750毫升去离子水（DH 2 O）;准备0.2M的索伦森缓冲液（pH 7.4）溶解13.8克氢化钠2 PO 4×1H 2 O和71.2克Na 2 HPO 4×2H 2 O在2.5L卫生署2 O. </li></ol></li><li> 脱水和嵌入 <ol><li>通过使用刀片切割组织成大约5毫米厚的部分。 </li><li>通过使用组织-Tek的贵宾真空渗入处理器（基于浸没入乙醇升序浓度的标准程序，随后二甲苯和最后石蜡（ 表1）开始所述组织硬化处理（脱水）。 </li><li>放置在组织中的模具和倒在液体石蜡的样品周围形成"石蜡块"。长期储存要求室温（RT）。然而，之前的切片，它可推荐以冷却块， 例如，过夜，在4℃（O / N）。</li></ol></li></ol> 2.切片<ol><li>放置石蜡块到雪橇切片机的固定支架，它可以在整个刀向前和向后移动。调整块和切片刀（这取决于刀的几何形状，而且还取决于切削速度和技术）之间的最佳角度。 </li><li>从石蜡块5微米厚截面 - 切3。 </li><li>转移只是削减部成一个水容器和从水随后把它安装到载玻片。 </li><li>小心地按安装段对纸巾去除残余的水和可能的气泡。市售预涂粘合剂载玻片是可取的。 </li><li>干幻灯片安装在炉灶的几个小时在50 - 60℃。 </li></ol> 3.脱蜡（补液和阻断内源性过氧化物酶） <ol><li>沉浸幻灯片2倍到二甲苯（或者替代二甲苯XEM-200），每次15 - 20&#39;。 </li><li>漂洗在99％的乙醇。 </li><li>要阻止内源性过氧化物酶的活性，在培养含0.25％过氧化氢甲醇溶液30&#39;的章节。 </li><li>通过使用乙醇随着水含量（99％，70％，在蒸馏水中结束了继续补液。 </li><li>从这个角度，直到盖的安装牍部分必须保持湿润。 </li></ol> 4.抗原修复<ol><li>使用蒸锅用于在抗原修复溶液沸腾的幻灯片（在此情况下的EDTA pH 8.5的缓冲液中）60&#39;（EDTA缓冲原液包含1.21克Tris和0.37克EDTA溶解在50毫升的dh 2 O;以制备工作解稀2.5毫升EDTA原液在47.5毫升卫生署2 O）。 </li><li>冷静下来RT上的幻灯片约。 1小时，然后漂洗3 - 5倍，用Tris缓冲盐水（TBS，使用可替换的PBS）组成的0.05M Tris和0.15M NaCl的的; pH值7.5调整无线日盐酸。或者利用商业TBS。 </li></ol> 5.阻断特异性结合位点<ol><li>为了避免非特异性的背景反应，在含有10％胎牛血清（FCS）和90％DAKO缓冲阻挡溶液孵育上RT下部分30&#39;。 </li></ol> 6.双击免疫标记:同时孵化与主阿布斯<ol><li>稀释的初级抗体的所需量在封闭液孵育在o / n 4℃。用α-CD68（ED1; 1:1000）:;为双免疫染色结合α-趋化因子（300 1 CCL11）或α-Iba1（AIF1，1:1000）或α-CD8α（OX-8; 1:200） 。 </li><li>冲洗幻灯片提升3至5用TBS缓冲液（或者使用稀释10倍缓冲DAKO）。 </li><li>在封闭溶液并在室温下孵育1小时:稀释的二抗的所需量（200生物素化的抗羊和AP缀合的抗小鼠，既1）。 </li><li>冲洗幻灯片3 - 5倍用TBS缓冲液（美国E或者10倍稀释DAKO缓冲）。 </li><li>孵育在对RT 1小时封闭溶液稀释生物素蛋白 - 辣根过氧化物酶复合物酶（HRP）的幻灯片。 </li><li>冲洗幻灯片3 - TBS缓冲液（或者使用稀释10倍缓冲DAKO）5倍。 </li></ol> 7.可视化<ol><li> 结合的AP标记的第二抗体的可视化 <ol><li>通过在1升的dh 2 O12.1克的Tris溶解制备的0.1M Tris-HCl缓冲液，并通过使用盐酸调节pH到。使用相同的Tris-HCl缓冲液，制备1M的左旋咪唑溶液。准备在卫生署2 O.新鲜4％的纳米2溶液</li><li>以获得将50ml坚牢蓝（FB）衬底（体积为一个标准的玻璃比色杯需要）溶解6.25毫克萘酚-AS-MX磷酸在玻璃管312.5微升DMF中并搅拌入50ml预热的（37 ℃）的Tris-HCl缓冲液中。在312.5微升2N盐酸溶解12.5毫克FB RR盐加以前312.5微升准备工作红4％的NaNO 2溶液，搅拌该混合物到相同50毫升温热预先的Tris-HCl缓冲液中。轻轻摇动，直到黄色液体变得清晰，最后加入77微升先前准备1M的左旋咪唑溶液中。滤液获得的混合物倒到放置在玻璃试管幻灯片。 </li><li>发起孵育在37℃并控制在光学显微镜下显影过程大约每15 - 30分钟。如果转向模糊，更换新鲜混合的FB的解决方案。 </li><li>冲洗幻灯片3 - 5倍用TBS缓冲和转移到随后PBS缓冲液。 </li></ol></li><li> 结合生物素化二级抗体的可视化 <ol><li>制备DAB / H 2 O 2稀释49毫升的PBS1毫升DAB原液（每1毫升的PBS 25毫克DAB）发展的解决方案。添加16.5微升H 2 O 2和滤液部分之前倒入。 </li><li>色原DAB转化成眉沉淀ñ颜料能够立竿见影。控制在光学显微镜下的发展过程（甚至是几秒钟长的潜伏期可能会引起高背景和屏蔽特定信号）。 </li><li>褐色色素沉淀物的强度可以通过孵育在包含2％的硫酸铜和0.9％的NaCl的5&#39;的溶液替代地增强。 </li><li>冲洗幻灯片3 - 5倍的PBS，最后与卫生署2 O，或者使用自来水。 </li><li>安装幻灯片有盖，直接从水中通过水GelTol安装介质滑落。避免产生气泡。 </li><li>允许安装介质的完全干燥， 例如，在o / n 4℃。商店干RT上的幻灯片。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1。 </td><td> ％乙醇</td><td> 20&#39; </td><td> 40℃ </td></tr><tr><td> 2。 </td><td> ％乙醇</td><td> 60＆＃39; </td><td> 40℃ </td></tr><tr><td> 3。 </td><td> ％乙醇</td><td> 90&#39; </td><td> 40℃ </td></tr><tr><td> 4。 </td><td> ％乙醇</td><td> 60&#39; </td><td> 40℃ </td></tr><tr><td> 5。 </td><td> ％乙醇</td><td> 90&#39; </td><td> 40℃ </td></tr><tr><td> 6。 </td><td> ％乙醇</td><td> 60&#39; </td><td> 40℃ </td></tr><tr><td> 7。 </td><td> ％乙醇</td><td> 90&#39; </td><td> 40℃ </td></tr><tr><td> 8。 </td><td> ％乙醇</td><td> 120&#39; </td><td> 40℃ </td></tr><tr><td> 9。 </td><td>二甲苯</td><td> 30&#39; </td><td> 40℃ </td></tr><tr><td> 10。 </td><td>二甲苯</td><td> 60&#39; </td><td> 40℃ </td></tr><tr><td> 11。 </td><td>石蜡</td><td> 60&#39; </td><td> 60℃ </td></tr><tr><td> 12。 </td><td>石蜡</td><td> 60&#39; </td><td> 60℃ </td></tr><tr><td> 13。 </td><td>石蜡</td><td> 60&#39; </td><td> 60℃ </td></tr><tr><td> 14。 </td><td>石蜡</td><td> 120&#39; </td><td> 60℃ </td></tr></tbody></table> 表1. 组织通过组织-Tek的VIP真空渗入Processor处理。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+endogenous+tissue+alkaline+phosphatase+with+the+use+of+alkaline+phosphatase+conjugates+in+immunohistochemistry.">Inhibition of endogenous tissue alkaline phosphatase with the use of alkaline phosphatase conjugates in immunohistochemistry.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 29, 981-984 (1981).</li><li>Petrelli, F., Coderoni, S., Moretti, P., Paparelli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+biotin+on+phosphorylation,+acetylation,+methylation+of+rat+liver+histones.">Effect of biotin on phosphorylation, acetylation, methylation of rat liver histones.</a> Molecular biology reports. 4, 87-92 (1978).</li><li>Yagi, T., Terada, N., Baba, T., Ohno, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+endogenous+biotin-containing+proteins+in+mouse+Bergmann+glial+cells.">Localization of endogenous biotin-containing proteins in mouse Bergmann glial cells.</a> The Histochemical journal. 34, 567-572 (2002).</li><li>Van Hecke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+Immunohistochemical+Staining+Today:+Choices+to+Make,+Challenges+to+Take.">Routine Immunohistochemical Staining Today: Choices to Make, Challenges to Take.</a> Journal of Histotechnology. 1, 45-54 (2002).</li></ol>

The authors declare no conflict of interest.

标准IHC程序往往需要具体的调整，以获得最佳的结果，这意味着通用丰富的经验，也有"试错"的方针。从组织制备直到目标的可视化，在协议中几乎每一步可经受为了提高最后结果单独设计的修改。这里提出双染色方案举例说明基于IHC蛋白靶向特别调整评估我们在受神经炎症福尔马林固定，石蜡包埋的大鼠中枢神经系统和LN组织切片感兴趣的目标蛋白质之间的相互关系。因此，它可以用作用于学习...

尽管先进的高通量的利用对甲基化，转录或甚蛋白质水平分析进行免疫染色仍然直接蛋白质检测的组织样品，细胞培养物或细胞涂片的黄金标准。通过揭示本地化/分布格局，免疫组织化学（IHC）可以评估的相对比例和靶蛋白的地形相互关系，甚至表明它们的生物活性。因此，IHC被广泛用于临床和研究的目的， 例如用于诊断，治疗评估，对疾病机制，在动物模型中的功能和表型的改变等研究基本上包含组织学，病理学，生物化学和免疫学，免疫组织化学已显著自1941年以来，当被用于在第一时间，以确定在受感染的组织1 肺炎球菌抗原的荧光标记抗体中高级。 V细胞产物和通过IHC组分isualization基于抗体（Abs）以下，以它们的特异性抗原（抗原）的结合。除了使用荧光团标记的抗体，免疫反应也可以通过使用酶像过氧化物2,3或碱性磷酸酶4可视化。此外，胶体金标记的抗体5被用于由两个光学和电子显微镜检测抗原特异性抗体的相互作用，而放射性标记物通过放射自显影显示。 该抗原 - 抗体免疫反应可以通过直接或间接方法进行检测。直接法本质上是更快，更简单，因为它使用直接标记主要腹肌6。然而，由于显著缺乏敏感性，间接方法是优选的直接的。两步间接检测程序需要未标记主腹肌，如在第一和标记的针对主绝对二次ABS，作为第二层7。信号放大可以通过进一步涉及实现，酶偶联叔抗体（三步间接法）结合所述次级抗体。常用的间接检测方法是亲和素 - 生物素和过氧化物酶antiperoxidase（PAP）。可替代地，碱性磷酸酶antialkaline磷酸酶（APAAP）复合物可以用于代替PAP法。值得注意的是，碱性磷酸酶（AP）的方法似乎是比免疫过氧化物酶方法4更敏感。抗生物素蛋白 - 生物素复合物（ABC）方法与任一标记的抗生物素蛋白 - 生物素复合物（LAB）组合使用生物素化的次级抗体，或标记的链霉抗生物素复合物（板）。检测灵敏度可进一步增加通过涉及用过氧化物酶标记或碱性磷酸酶8抗生物素蛋白。在使用其他检测方法是聚合物标签，酪胺放大和免疫滚圈9。值得注意的是，不同的检测方法可以组合在同一组织的多个银检测样品，将其在这里提出1978年4。同时双免疫染色报道首次用过氧化物酶-结合的和AP偶联的二抗，分别在福尔马林固定，石蜡包埋的大鼠中枢神经系统和LN组织切片进行。信号用3,3&#39;-二氨基联苯胺（DAB）色原体和固蓝（FB）APAAP复杂，分别显现。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Isoflurane (Isofluran): 1-Chlor-2,2,2-trifluorethyl (difluormethyl ether) 2-Chlor-2-(difluormethoxy)-1,1,1-trifluorethan (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;ClF&lt;sub&gt;5&lt;/sub&gt;O)</td><td>Baxter</td><td>1001936060</td><td>Eye irradiation. Probably influences fertility and damages baby in uterus. Administer only in adequately equipped anesthetizing environment.</td></tr><tr><td>Sodiumchloride (NaCl)</td><td>Merck</td><td>1.0604</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS), tablet</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>Paraformaldehyde (OH(CH&lt;sub&gt;2&lt;/sub&gt;O)&lt;sub&gt;n&lt;/sub&gt;H (n = 8 - 100)), 4% in 1x PBS</td><td>Apl pharma</td><td>34 24 28</td><td>Health hazard. Corrosive. Flamable. Acute toxicity.</td></tr><tr><td>Ethanol &amp;#8805;99.5%, absolute (CH&lt;sub&gt;3&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;OH)</td><td>Sigma-Aldrich</td><td>459844-1L</td><td>Flamable.</td></tr><tr><td>Xylene (Xylenes, histological grade; C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;(CH&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt; </td><td>Sigma-Aldrich</td><td>534056</td><td>Flamable. Acute toxicity.</td></tr><tr><td>Histo-Comp Paraffin Wax</td><td>Tissue-Tek</td><td>V0-5-1001</td><td></td></tr><tr><td>Adhesive Microscope Slides</td><td>Starfrost</td><td>MIC-1040-W</td><td></td></tr><tr><td>Xylol substitute XEM-200 </td><td>Vogel GmbH</td><td>ND-HS-200</td><td>Respiratory sensitization. Carcinogenicity.</td></tr><tr><td>&amp;alpha;-CD8 (Ox-8) mouse anti-rat, primary antibody</td><td>AbD Serotec</td><td>MCA48G</td><td></td></tr><tr><td>&amp;alpha;-Iba1 (AIF1) mouse anti-rat, primary antibody</td><td>Millipore</td><td>MABN92</td><td></td></tr><tr><td>&amp;alpha;-CD68 (ED1) mouse anti-rat, primary antibody</td><td>AbD Serotec</td><td>MCA341R</td><td></td></tr><tr><td>&amp;alpha;-eotaxin C-19 (CCL11) goat anti-rat, primary antibody</td><td>Santa Cruz BT</td><td>SC-6181</td><td></td></tr><tr><td>Alkaline phosphatase (AP)-conjugated secondary antibody</td><td>Dakopatts, Denmark</td><td>D0314</td><td></td></tr><tr><td>Biotinylated secondary antibody</td><td>Amersham Biotech</td><td>RPN1025</td><td></td></tr><tr><td>Avidin- horseradish peroxidase complex (HRP) </td><td>Sigma-Aldrich</td><td>A3151</td><td></td></tr><tr><td>Naphthol AS-MX phosphate (C&lt;sub&gt;19&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;NO&lt;sub&gt;5&lt;/sub&gt;P)</td><td>Sigma-Aldrich</td><td>N4875-1G</td><td>Acute toxicity.</td></tr><tr><td>Fast Blue RR Salt, Azoic Diazo No. 24 (C&lt;sub&gt;15&lt;/sub&gt;H&lt;sub&gt;14&lt;/sub&gt;ClN&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt; x 1/2 ZnCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>FBS25</td><td></td></tr><tr><td>Levamisol hydrochloride (C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;13&lt;/sub&gt;ClN&lt;sub&gt;2S&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>31742</td><td>Acute toxicity.</td></tr><tr><td>3,3&#039;-Diaminobenzidine tetrahydrochloride (DAB Chromogen)</td><td>DAKO</td><td>S3000</td><td>Highly flammable. Toxic.</td></tr><tr><td>Copper sulphate (CuSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Merck</td><td>1.02791</td><td>Acute toxicity. Environmental hazzard.</td></tr><tr><td>GelTol Aqueous Mounting Medium</td><td>Thermo Electron Corporation</td><td>230100</td><td></td></tr><tr><td>Hydrogen peroxide, 30% (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Merck</td><td>107210</td><td>Acute toxicity.</td></tr><tr><td>Methanol (CH&lt;sub&gt;3&lt;/sub&gt;OH)</td><td>Fluka</td><td>65543</td><td>Acute toxicity. Respiratory sensitization. Carcinogenicity. Flammable.</td></tr><tr><td>Tris (hydroxymethyl) aminomethane, TRIS base (C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt; )</td><td>AppliChem</td><td>A1379</td><td>Skin and eye irritation.</td></tr><tr><td>Tris Buffered Saline (TBS), tablet</td><td>Sigma-Aldrich</td><td>T5030</td><td></td></tr><tr><td>Di- Sodium hydrogen phosphate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Merck</td><td>1.0658</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; x 1H&lt;sub&gt;2&lt;/sub&gt;O) </td><td>Merck</td><td>1.06346</td><td></td></tr><tr><td>Fetal calf serum (FCS) </td><td>Cambrex BioScience</td><td>DE-14-802F</td><td></td></tr><tr><td>DAKO cytomation wash buffer 10x </td><td>DAKO</td><td>S3006</td><td>Should be stored at 2-8 &amp;deg;C to inhibit bacterial growth. Avoid foaming.</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide; DMF (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;7&lt;/sub&gt;NO)</td><td>Fluka</td><td>40250</td><td>Flammable. Acute toxicity.</td></tr><tr><td>Glas coverslips 24 x 36 mm </td><td>Menzel-Gl&amp;#228;ser</td><td>BB024036A1</td><td></td></tr><tr><td>Hydrochloric acid, conc. (HCl)</td><td>Sigma-Aldrich</td><td>30721</td><td>Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic.</td></tr><tr><td>Hydrochloric acid solution volumetric, 2 M HCl (2 N)</td><td>Fluka</td><td>71826</td><td>Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic.</td></tr><tr><td>Sodium nitrite, ReagentPlus, &amp;#8805;99.0% (NaNO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S2252</td><td>Oxidant. Toxic. Dangerous for the environment.</td></tr><tr><td>Ethylenedinitrilotetraacetic acid disodium salt dihydrate (EDTA; C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;14&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;Na&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Merck</td><td>1.08454</td><td>Oral exposures may cause reproductive and developmental effects.</td></tr><tr><td colspan="4">&lt;strong&gt; Equipment&lt;/strong&gt;</td></tr><tr><td>Tissue-Tek&lt;sup&gt;V.I.P&lt;/sup&gt; Vacuum Infiltration Processor</td><td>Sakura</td><td>5902 VIP Jr. 115 V, 60 Hz</td><td></td></tr><tr><td>Hacker-Bright 8000 Series Base Sledge Microtome </td><td>Hacker instruments</td><td></td><td></td></tr><tr><td>Household food steamer </td><td>Braun</td><td>MultiGourmet FS 20</td><td></td></tr><tr><td>Light microscope </td><td>Leica Polyvar 2</td><td></td><td></td></tr></tbody></table>

immunohistochemical analysis in the rat central nervous system and peripheral lymph node tissue sections

免疫组织化学（IHC）提供了非常具体的，可靠的和有吸引力的蛋白质可视化。基于IHC-多色标记的正确性能和解释是具有挑战性的，特别是当用于在具有高脂肪含量的组织评估靶蛋白之间的相互关系，如对中枢神经系统（CNS）。 我们的协议代表特别调整为在大鼠中枢神经系统和受神经炎症外周淋巴结（LN）的检测结构和可溶性蛋白的标准免疫标记技术的改进。尽管如此，有或没有进一步的修改，我们的协议可以有可能被用于检测的其它相关蛋白质的目标，即使是在其它器官和物种比这里提出。

双immunostainings（共染色）的福尔马林固定，石蜡包埋的鼠的CNS和LN切片进行。使用雪撬切片3-5微米厚的组织切片切，随后安装到预涂布粘合剂载玻片并如前所述10,11,12处理。简言之，脱石蜡，组织再水化和内源性过氧化物失活后，切片进行抗原修复过程，随后封闭步骤以消除非特异性结合位点。共染色是在下列组合进行:ⅰ）CCL11 /伊巴-1，ⅱ）CCL11 / ED1和iii）CCL11 / CD...

Watch this Scientific Journal Video about Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections at JoVE.com

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

immunohistochemical-analysis-rat-central-nervous-system-peripheral

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.0K 次观看.  Karolinska Institutet. We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

视频: Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

淋巴结冷冻科肿瘤细胞粘附的定量

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...

科研

癌症研究

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

引物对免疫冰冻切片大鼠脑组织:例为染色小胶质细胞和神经元

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

神经科学

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

对于神经化学标记用biocytin填充和加工部分的免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

免疫与感染

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

佩尔维奇自主性甘利亚与雄性大鼠和雌性大鼠相关神经的解剖

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To maintain the morphology of these delicate tissues, it is routine to administer a chemical fixative such as paraformaldehyde via cannulation of the heart in anesthetized animals (transcardial perfusion). Transcardial perfusion of the mouse heart has traditionally relied on the use of peristaltic pumps or air pressure to deliver both the saline and fixative solutions necessary for this process. As an easily accessible alternative to these methods, this work demonstrates the use of a gravity-fed method of perfusate delivery that uses materials available in most hardware stores. 
To validate this new perfusion method, this work demonstrates all the subsequent steps necessary for the sensitive detection of phosphorylated &#945;-synuclein in both the brain and spinal cord. Included in these steps are the dissection of the fixed brain and spinal cord tissues, rapid freezing/embedding and cryosectioning of the tissues, and immunofluorescent staining. As this method results in whole-body delivery of the fixative, it may also be used to prepare other non-neuronal tissues for histologic analysis.

A convenient gravity-fed perfusion method for histological analysis of the mouse central nervous system is presented. Immunofluorescent detection of phosphorylated &#945;-synuclein is demonstrated in a mouse model of Parkinson's disease. This work also comprehensively describes the transcardial perfusion, dissection, tissue freezing/embedding, and frozen sectioning steps.

一种重力喂养的经心灌注方法，用于小鼠中枢神经系统的组织学分析

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To ...

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

使用自由浮动组织切片进行基于组织学的染色

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

生物学

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Source: Singh, P., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/60405">Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020)
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