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 border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Isoflurane 			(Isofluran): 1-Chlor-2,2,2-trifluorethyl (difluormethyl 			ether) 
 2-Chlor-2-(difluormethoxy)-1,1,1-trifluorethan 			(C3H2ClF5O)</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(CH2O)nH (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 			&ge;99.5%, absolute (CH3CH2OH)</td>
 <td>Sigma-Aldrich</td>
 <td>459844-1L</td>
 <td>Flamable.</td>
 </tr>
 <tr>
 <td>Xylene 			(Xylenes, histological grade; C6H4(CH3)2 </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>&alpha;- CD8 			(Ox-8) mouse anti-rat, primary antibody</td>
 <td>AbD 			Serotec</td>
 <td>MCA48G</td>
 <td></td>
 </tr>
 <tr>
 <td>&alpha;- Iba1 			(AIF1) mouse anti-rat, primary antibody</td>
 <td>Millipore</td>
 <td>MABN92</td>
 <td></td>
 </tr>
 <tr>
 <td>&alpha;- CD68 			(ED1) mouse anti-rat, primary antibody</td>
 <td>AbD 			Serotec </td>
 <td>MCA341R</td>
 <td></td>
 </tr>
 <tr>
 <td>&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 (C19H18NO5P)</td>
 <td>Sigma-Aldrich</td>
 <td>N4875-1G</td>
 <td>Acute 			toxicity.</td>
 </tr>
 <tr>
 <td>Fast Blue 			RR Salt, Azoic Diazo No. 24 (C15H14ClN3O3 			x 			1/2 ZnCl2)</td>
 <td>Sigma-Aldrich</td>
 <td>FBS25</td>
 <td></td>
 </tr>
 <tr>
 <td>Levamisol 			hydrochloride (C11H13ClN2S)</td>
 <td>Sigma-Aldrich</td>
 <td>31742</td>
 <td>Acute 			toxicity.</td>
 </tr>
 <tr>
 <td>3,3'-Diaminobenzidine 			tetrahydrochloride (DAB Chromogen)</td>
 <td>DAKO</td>
 <td>S3000 </td>
 <td>Highly 			flammable. Toxic.</td>
 </tr>
 <tr>
 <td>Copper 			sulphate (CuSO4)</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% (H2O2)</td>
 <td>Merck</td>
 <td>107210</td>
 <td>Acute 			toxicity.</td>
 </tr>
 <tr>
 <td>Methanol 			(CH3OH)</td>
 <td>Fluka</td>
 <td>65543</td>
 <td>Acute 			toxicity. Respiratory sensitization. Carcinogenicity. Flammable.</td>
 </tr>
 <tr>
 <td>Tris 			(hydroxymethyl) aminomethane, TRIS base (C4H11NO3 			)</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 (Na2HPO4 			x 			2H2O)</td>
 <td>Merck</td>
 <td>1.0658</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium 			dihydrogen phosphate monohydrate (NaH2PO4 			x 			1H2O) </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 &deg;C to inhibit bacterial growth. Avoid foaming.</td>
 </tr>
 <tr>
 <td>N,N- 			Dimethylformamide; DMF (C3H7NO)</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&auml;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, 2M HCl (2N) </td>
 <td>Fluka </td>
 <td>71826</td>
 <td>Corrosive, 			irritant, permeator. Lung sensitizer (as acid mist). Toxic.</td>
 </tr>
 <tr>
 <td>Sodium 			nitrite, ReagentPlus, &ge;99.0% (NaNO2)</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; C10H14N2Na2O8 			x 2H2O)</td>
 <td>Merck</td>
 <td>1.08454</td>
 <td>Oral 			exposures may cause reproductive and developmental effects.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td> Equipment</td>
 </tr>
 <tr>
 <td>Tissue-Tek 			V.I.P 			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

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

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断:一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

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