整装在鸡的抗体染色方法
Summary
本视频演示了整个安装免疫组织化学，抗原的时空表达模式的方法是，可以在年轻的鸡胚可视。这种方法最初是由简多德和汤姆Jessell。
Abstract
鸡胚是一个有价值的工具，在早期胚胎发育的研究。其透明度，可获得性和易于操纵，使其研究在发育中的大脑，神经管和体节抗体表达的理想工具。本视频演示了在整个安装使用HRP标记二抗抗体染色的不同步骤，首先，从鸡蛋解剖胚胎和多聚甲醛固定。二，内源性过氧化物酶的灭活胚胎，然后暴露的主要抗体。经过多次洗涤，胚胎培养，以辣根过氧化物酶标记的第二抗体。过氧化物酶活性，发现用二氨基联苯胺底物的反应。最后，胚胎是固定的，摄影和切片处理。在使用荧光抗体这种方法的优点是可以处理的胚胎蜡切片，从而使截面抗原的网站研究。这种方法最初是由简多德和汤姆Jessell<sup> 1</sup>。
Protocol
一，原理概述： 本视频演示了在整个装载在鸡胚免疫组织化学的不同步骤。首先，胚胎是固定在煤灰[IHC1]。然后淬火，内源性过氧化物酶的活性[IHC2]。胚胎培养中的主要抗体[IHC3]。经过多次洗涤，胚胎是在二抗孵育[IHC4];显色反应显示使用民建联[IHC5并出现抗体染色橙[IHC6]。 <p cla…
Discussion
本视频演示了年轻的鸡胚在执行整个安装抗体染色的不同步骤。这个协议基本上是用于小鸡 2,3表征的新型抗体的空间和时间，以及已知的抗原标志物的使用，以确定胚胎畸形，侮辱4。
Acknowledgements
取自发育研究杂交瘤细胞NICHD的和维护由美国爱荷华大学生物科学系的主持下开发银行（J.多德，TM Jessell和A.川）开发的单克隆抗体（15.3B9，PAX7） ，爱荷华州。 DP是从国家药物滥用研究所露丝Kirschstein奖1F32 DA021977 – 01A1收件人。这项工作是支持的玛格丽特M. Alkek基金会RHF。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Eggs | Charles River Laboratories | Premium Fertile | Fertilized, HH4 (16 hr) | |
| Stereomicroscope | Microscope | Leica Microsystems | MZ9.5 or similar | |
| Marsh Automatic Incubator | 기타 | Lyon | RX | |
| Curved Forceps (1) | Tool | Electron Microscopy Sciences | 72991-4C | |
| Forceps (2) | Tool | Fine Science Tools | 11002-13 | |
| Fine scissors | Tool | Fine Science Tools | 14161-10 | |
| Plastic dishes | Tool | Falcon | 353001 | |
| Rubber Bulb | Tool | Electron Microscopy Sciences | 70980 | |
| Pasteur Capillary Pipette | Tool | Electron Microscopy Sciences | 70950-12 | round edge under flame |
| Microdissecting knife | Tool | Fine Science Tools | 10056-12 | Use to cut embryo from surrounding membranes following fixation |
| Sylgard 184 Silicon Elastomer Curing Agent and Base | Reagent | Dow Corning | 0001986475 | Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C |
| 16% PFA | Reagent | Electron Microscopy Sciences | 15710 | |
| 30% H2O2 | Reagent | Sigma | H1009 | |
| BSA | Reagent | Sigma | A3803 | |
| NGS | Reagent | Jackson Immuno | 005-000-121 | |
| Primary Antibodies | Reagent | Developmental studies Hybridoma Bank | 4G11, 15.3B9, PAX7 | For these antibodies, investgators used mouse donnors |
| Peroxidase conjugated-Goat Anti-Mouse IgG (H+L) | Reagent | Jackson Immuno | 115-035-003 | |
| 3,3’-diaminobenzidine tetrahydrochloride | Reagent | Pierce | 34001 | Store at -20; Allow bottle to warm to RT before use. |
| Cedar wood oil | Reagent | Sigma | W522503 | |
| Fast green FCF | Reagent | Sigma | F7252 | |
| Minuten pins 0.2mm diam | Fine Science Tools | 26002-20 |
References
- Yamada, T., Placzek, M., Tanaka, H., Dodd, J., Jessell, T. M. Control of cell pattern in the developing nervous system: Polarizing activity of the floor plate and notochord. Cell. 64, 635-647 (1991).
- Streit, A., Stern, C. D., Thery, C., Ireland, G. W., Aparicio, S., Sharpe, M. J., Gherardi, E. A role for HGF/SF in neural induction and its expression in Hensen’s node during gastrulation. Development. 121, 813-824 (1995).
- Basch, M., Bronner-Fraser, M., Garcia-Castro, M. Specification of neural crest occurs during gastrulation and requires Pax7. Nature. 441, 218-222 (2006).
- Psychoyos, D., Stern, C. D. Restoration of the organizer after radical ablation of Hensen’s node and the anterior primitive streak in the chick embryo. Development. 122, 3263-3273 (1996).
Tags
Whole Mount Antibody StainingChick EmbryoEarly Embryonic DevelopmentAntibody ExpressionDeveloping BrainNeural TubeSomiteHRP Conjugated Secondary AntibodiesParaformaldehyde FixationInactivation Of Endogenous PeroxidasePrimary Antibody ExposureWashesSecondary Antibody IncubationPeroxidase ActivityDiaminobenzidine Substrate ReactionWax SectioningAntigen SitesJane DoddTom Jessell
Cite This Article
Psychoyos, D., Finnell, R. Method for Whole Mount Antibody Staining in Chick. J. Vis. Exp. (24), e956, doi:10.3791/956 (2009).