免疫组化和多标签与同一宿主物种抗体研究成人神经发生
Summary
This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.
Abstract
成人神经发生是在其从激活的神经干细胞通过越来越致力于中间祖亚型产生新的神经元一高度调节，多阶段的过程。每个这些亚型的表达的一组，连同具体形态学标准，可以用于其识别特定的分子标志物。典型地，免疫荧光技术应用于涉及亚型特异性抗体结合外切或内源性增殖标记。在此我们描述免疫标记成人海马神经发生的各个阶段的检测和定量的方法。这些包括胸苷类似物，穿心灌注，组织处理，热诱导抗原活化，ABC免疫组织化学，多个间接免疫荧光，激光共聚焦显微镜和细胞量化的应用。此外我们提出了一个连续的多个免疫协议，规避问题ü所产生的由使用在相同的宿主物种中产生的第一抗体的需要sually。它允许一个准确的识别所有海马祖亚型连同在一个单一的部分扩散标记。这些技术是一种强大的工具，研究不同祖亚型并联的调节，它们在脑病理学的参与以及它们在特定大脑功能的作用。
Introduction
两个脑区的组成产生整个人生新的神经元，侧脑室的脑室下区和海马齿状回（DG）的颗粒下区（SGZ）。新生神经元的神经祖细胞获得茁壮1,2之前通过形态和生理发育的不同阶段。从缓慢除以径向胶质样干细胞（1型）的转运连续阶段扩增中间祖细胞产生。更未分化的亚型（式2a和式2b）中具有不规则的形状，总之，切向流程。它们所产生的神经母细胞（类型3），该逐步退出细胞周期成为未成熟神经元（与树突伸向分子层），最后整合进海马网络作为成熟粒细胞。由于其特殊的生理特点，这些细胞提供电路具有增强的可塑性3 sugges婷在海马功能独特的作用。事实上，在过去十年的研究产生的大量证据表明，成年神经有助于空间记忆，模式分离和情感行为4,5。
成年神经可以用不同的方法进行研究。胸苷类似物掺入到DNA中的S期的细胞周期，并允许新生细胞6-8出生约会，定量和命运分析。不同胸苷类似物（ 例如，CldU，埃杜或IDU）的顺序应用可以用于研究细胞周转或实验9的过程中，出生在不同时间点的细胞群。另一种，内源性标志物细胞增殖Ki67的。它表达于在细胞周期（G1，S，G2，M），除休止期（G0）及G1 10,11的开始的各个阶段的分裂细胞。分析新生细胞群的表型的成人dentat在Ë回几个阶段特异性的分子标记可用于如GFAP，巢蛋白，DCX和的NeuN 1,6。 GFAP是星形胶质细胞成熟的标志，但也表达了放射状胶质细胞样细胞在成人脑。巢蛋白是特异的放射状胶质细胞样细胞和早期中间祖细胞的中间丝。 DCX是表示在中间祖细胞，神经母细胞和未成熟的神经元微管相关蛋白。基于这三个标记和标记的细胞四个不同的祖细胞的亚型可以识别的形态特征的（共）表达：1型（GFAP +，巢蛋白+，DCX – ），2a型（GFAP – ，巢蛋白+ ，DCX – ），2B型（GFAP – ，+巢，DCX +）和3型（GFAP – ，巢– ，DCX +）1。 DCX用的NeuN，这表现在有丝分裂后的神经元，一起共同标签允许immatur分化E（DCX +，+的NeuN）和成熟（DCX – ，+的NeuN）颗粒神经元。
上面提到的标记物经常用于免疫荧光共标记和随后的共聚焦显微镜来分析新生细胞的数目和身份。这通常需要来自不同宿主物种的抗体，以防止不希望的抗体交叉反应性。然而，大多数适于神经发生研究初级抗体被升高或者家兔或小鼠（ 例如，小鼠α-BrdU的小鼠α-的NeuN，兔α-Ki67的，兔α-GFAP）。这导致以可能在一个单一的切片进行评估抗原的数量和组合严重的局限性。这反过来不仅增加了染色的努力，因为多种染色必须执行，而且还可能影响结果的可靠性。此外，一些抗原易受福尔马林固定诱导抗原掩蔽（ 例如 Ki67的，巢）。我们在此从古典单和多协议免疫标记描述修改（ 如表位检索，多个连续染色，使用巢蛋白GFP转基因小鼠12）克服许多问题。特别是，连续的多个免疫协议允许对染色即使抗体的一部分从同一主机衍生多达四个不同的抗原。这使得能够同时检测1型，图2a，2b型和3型祖细胞，以及在一个单一的部分其增殖活性。
Protocol
Representative Results
Discussion
量化和鉴定新生细胞亚群是在成年神经研究的一个核心问题。结合增殖标记和抗体在成年神经的特定阶段表达的蛋白质可以让免疫组化检测这些亚群。一些抗体或抗体组合的需要特定的染色条件。
分裂细胞合成胸腺嘧啶核苷类似物的标记仍然是金标准研究成年海马神经发生。关键的是要考虑到开始实验前适当注射协议。我们通常施用50毫克/千克（体重，IP）的BrdU，但浓度不?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank S. Tausch for excellent technical assistance. The work was supported by BMBF (Bernstein Focus 01GQ0923) and DFG (FOR1738).
Materials
| Name | Company | Catalog Number | Comments & Dilutions |
| Thymidine analog administration | |||
| 5-Bromo-2′-deoxyuridine, BrdU | Sigma-Aldrich | B9285 | toxic (mutagenic, teratogenic) |
| 5-Chloro-2′-deoxyuridine, CldU | Sigma-Aldrich | C6891 | |
| 5-Chloro-2′-deoxyuridine, CldU | MP Biomedicals | 2105478 | |
| 5-Iodo-2′-deoxyuridine, IdU | MP Biomedicals | 2100357 | |
| Tissue preparation | |||
| Isoflurane-Actavis | Piramal Healthcare | 700211 | |
| Paraformaldehyde powder (PFA) | Riedel-De Häen | 16005 | toxic, flammable |
| Perfusion pump PD5206 | Heidolph Instruments | 523-52060-00 | |
| Masterflex Tygon lab tubing, Ø 0.8 mm | Thermo Fischer Scientific | 06409-13 | |
| Feeding needle, straight, 21G, 1.75mm olive tip, 40mm | Agnthos | 1036 | |
| Freezing microtome Microm HM 400 | Thermo Fischer Scientific | ||
| 24 Well Cell Culture Multiwell Plates | Greiner Bio-One | 662160 | |
| Immunohistochemistry | |||
| Tefal Vitacuisine Steamer | Tefal | VS 4001 | |
| Netwell 24mm Polyester Mesh Membrane Inserts Pre-Loaded in 6-Well Culture Plates | Corning | 3479 | |
| Netwell 15mm Polyester Mesh Membrane Inserts Pre-Loaded in 12-Well Culture Plates | Corning | 3477 | |
| Netwell Plastic 6-Well Carrier Kit for 24mm Polyester Mesh Membrane Inserts | Corning | 3521 | |
| Netwell Plastic 12-Well Carrier Kit for 15mm Polyester Mesh Membrane Inserts | Corning | 3520 | |
| Vectastain Elite ABC Kit | Vector Laboratories | PK-6100 | |
| DAB (3,3′-Diaminobenzidine tetrahydrochloride hydrate) | Sigma-Aldrich | D-5637 | carcinogenic, light sensitive |
| Fluoromount-G | SouthernBiotech | 0100-01 | |
| Primary antibodies | |||
| rabbit IgG1 α-Ki67 | Novocastra/ Leica Biosystems | NCL-L-Ki67MM1 | DAB 1:400/IF 1:100; requires epitope retrieval |
| rabbit α-GFAP, AS-3-GF | Synaptic Systems | 173 002 | 1:500 |
| goat IgG (H+L) α-GFP | Acris Antibodies | R1091P | 1:300 |
| mouse IgG1 α-nestin | Abcam | ab6142 | 1:200; requires epitope retrieval |
| guinea pig IgG (H+L) α-Doublecortin | Merck Millipore | AB2253 | 1:500 |
| rat IgG2a α-BrdU (ascites) | AbD Serotec/ Bio-Rad | OBT0030CX | for detection of BrdU; DAB 1:500/IF 1:400 |
| rat IgG2a α-BrdU (purified) | AbD Serotec/ Bio-Rad | OBT0030 | for detection of CldU; DAB 1:500/IF 1:250-400 |
| mouse IgG1ĸ α-BrdU | BD Biosciences | 347580 | for detection of IdU; DAB 1:500/IF 1:350 |
| mouse IgG1 α-NeuN | Merck Millipore | MAB377 | 1:500 |
| Secondary antibodies | |||
| donkey α-guinea pig IgG (H+L)-Biotin | Dianova | 711-065-152 | 1:500 |
| donkey α-rat IgG (H+L)-Biotin | Dianova | 712-065-150 | 1:500 |
| donkey α-mouse IgG (H+L)-Biotin | Dianova | 715-065-151 | 1:500 |
| goat α-rat IgG (H+L)-Alexa Fluor 488 | Molecular Probes | A11006 | 1:250 |
| donkey α-goat IgG (H+L)-Alexa Fluor 488 | Molecular Probes | A11055 | 1:250 |
| donkey α-mouse IgG (H+L)-FITC, Fab-Fragment | Dianova | 715-097-003 | 1:100 |
| donkey α-mouse IgG (H+L)-Alexa Fluor 647 | Dianova | 715-605-151 | 1:250 |
| donkey α-guinea pig IgG (H+L)-Alexa Fluor 647 | Dianova | 706-605-148 | 1:250 |
| donkey α-rat IgG (H+L)-Rhodamine Red-X | Dianova | 712-295-150 | 1:250 |
| donkey α-rabbit IgG (H+L)-Rhodamine Red-X | Dianova | 711-295-152 | 1:250 |
| donkey α-guinea pig IgG (H+L)-Rhodamine Red-X | Dianova | 706-296-148 | 1:250 |
| Streptavidin-Rhodamine Red-X | Dianova | 016-290-084 | 1:500 |
| goat α-rabbit IgG (H+L)-AMCA | Dianova | 111-155-144 | 1:250, works only with rabbit α-GFAP |
| Hoechst 33342 | Molecular Probes | H3570 | 1:1000 |
| DAPI | Molecular Probes | D1306 | 1:1000 |
| Blocking | |||
| Fab-fragment donkey α-mouse IgG (H+L) | Dianova | 715-007-003 | 1:20 |
| Fab-fragment donkey α-rabbit IgG (H+L) | Dianova | 711-007-003 | 1:20 |
| Normal donkey serum | Merck Millipore | S30 | |
| Normal rabbit serum | Dianova | 011-000-010 | |
| Normal goat serum | Dianova | 005-000-001 | |
| Bovine Serum Albumine | Sigma-Aldrich | A7906 | |
| Histology | |||
| Cresyl violett | Sigma-Aldrich | C5042 | |
| Neo-Clear | Merck Millipore | 109843 | non-toxic xylene substitute |
| Neo-Mount | Merck Millipore | 109016 | permanent mounting medium |
| Microscopy | |||
| Axioskop 2 | Carl Zeiss Microscopy | ||
| LSM 710 | Carl Zeiss Microscopy |
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