免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片
概要
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.
Abstract
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。
Introduction
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'-二氨基联苯胺（DAB）色原体和固蓝（FB）APAAP复杂，分别显现。
Protocol
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 灌注和固定 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％?…
Representative Results
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 / …
Discussion
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?…
開示
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Reagent | |||
| Isoflurane (Isofluran): 1-Chlor-2,2,2-trifluorethyl (difluormethyl ether) 2-Chlor-2-(difluormethoxy)-1,1,1-trifluorethan (C3H2ClF5O) | Baxter | 1001936060 | Eye irradiation. Probably influences fertility and damages baby in uterus. Administer only in adequately equipped anesthetizing environment. |
| Sodiumchloride (NaCl) | Merck | 1.0604 | |
| Phosphate Buffer Saline (PBS), tablet | Sigma-Aldrich | P4417 | |
| Paraformaldehyde (OH(CH2O)nH (n = 8 – 100), 4% in 1x PBS | Apl pharma | 34 24 28 | Health hazard. Corrosive. Flamable. Acute toxicity. |
| Ethanol ≥99.5%, absolute (CH3CH2OH) | Sigma-Aldrich | 459844-1L | Flamable. |
| Xylene (Xylenes, histological grade; C6H4(CH3)2 | Sigma-Aldrich | 534056 | Flamable. Acute toxicity. |
| Histo-Comp Paraffin Wax | Tissue-Tek | V0-5-1001 | |
| Adhesive Microscope Slides | Starfrost | MIC-1040-W | |
| Xylol substitute XEM-200 | Vogel GmbH | ND-HS-200 | Respiratory sensitization. Carcinogenicity. |
| α- CD8 (Ox-8) mouse anti-rat, primary antibody | AbD Serotec | MCA48G | |
| α- Iba1 (AIF1) mouse anti-rat, primary antibody | Millipore | MABN92 | |
| α- CD68 (ED1) mouse anti-rat, primary antibody | AbD Serotec | MCA341R | |
| α- eotaxin C-19 (CCL11) goat anti-rat, primary antibody | Santa Cruz BT | SC-6181 | |
| Alkaline phosphatase (AP)-conjugated secondary antibody | Dakopatts, Denmark | D0314 | |
| Biotinylated secondary antibody | Amersham Biotech | RPN1025 | |
| Avidin- horseradish peroxidase complex (HRP) | Sigma-Aldrich | A3151 | |
| Naphthol AS-MX phosphate (C19H18NO5P) | Sigma-Aldrich | N4875-1G | Acute toxicity. |
| Fast Blue RR Salt, Azoic Diazo No. 24 (C15H14ClN3O3 x 1/2 ZnCl2) | Sigma-Aldrich | FBS25 | |
| Levamisol hydrochloride (C11H13ClN2S) | Sigma-Aldrich | 31742 | Acute toxicity. |
| 3,3′-Diaminobenzidine tetrahydrochloride (DAB Chromogen) | DAKO | S3000 | Highly flammable. Toxic. |
| Copper sulphate (CuSO4) | Merck | 1.02791 | Acute toxicity. Environmental hazzard. |
| GelTol Aqueous Mounting Medium | Thermo Electron Corporation | 230100 | |
| Hydrogen peroxide, 30% (H2O2) | Merck | 107210 | Acute toxicity. |
| Methanol (CH3OH) | Fluka | 65543 | Acute toxicity. Respiratory sensitization. Carcinogenicity. Flammable. |
| Tris (hydroxymethyl) aminomethane, TRIS base (C4H11NO3 ) | AppliChem | A1379 | Skin and eye irritation. |
| Tris Buffered Saline (TBS), tablet | Sigma-Aldrich | T5030 | |
| Di- Sodium hydrogen phosphate dihydrate (Na2HPO4 x 2H2O) | Merck | 1.0658 | |
| Sodium dihydrogen phosphate monohydrate (NaH2PO4 x 1H2O) | Merck | 1.06346 | |
| Fetal calf serum (FCS) | Cambrex BioScience | DE-14-802F | |
| DAKO cytomation wash buffer 10x | DAKO | S3006 | Should be stored at 2-8 °C to inhibit bacterial growth. Avoid foaming. |
| N,N- Dimethylformamide; DMF (C3H7NO) | Fluka | 40250 | Flammable. Acute toxicity. |
| Glas coverslips 24 x 36 mm | Menzel-Gläser | BB024036A1 | |
| Hydrochloric acid, conc. (HCl) | Sigma-Aldrich | 30721 | Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic. |
| Hydrochloric acid solution volumetric, 2M HCl (2N) | Fluka | 71826 | Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic. |
| Sodium nitrite, ReagentPlus, ≥99.0% (NaNO2) | Sigma-Aldrich | S2252 | Oxidant. Toxic. Dangerous for the environment. |
| Ethylenedinitrilotetraacetic acid disodium salt dihydrate (EDTA; C10H14N2Na2O8 x 2H2O) | Merck | 1.08454 | Oral exposures may cause reproductive and developmental effects. |
| Equipment | |||
| Tissue-Tek V.I.P Vacuum Infiltration Processor | Sakura | 5902 VIP Jr. 115 V, 60 Hz | |
| Hacker-Bright 8000 Series Base Sledge Microtome | Hacker instruments | ||
| Household food steamer | Braun | MultiGourmet FS 20 | |
| Light microscope | Leica Polyvar 2 |
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