双荧光原位杂交食脑切片
Summary
该协议涉及非放射性<em>原位</em>杂交程序，使在脊椎动物的大脑薄片，同时识别两个种成绩单，在一个单细胞的决议。
Abstract
在这里，我们描述了一个双荧光修改后的版本<em>原位</em优化杂交（dFISH）方法检测mRNA的新鲜冰冻脑切片的兴趣。我们的小组已经成功地使用这种方法，研究基因的共同监管。更具体地说，我们使用的dFISH方法，探讨组织的解剖，神经化学物质的属性，和感官体验在中央感官电路的影响，在单细胞分辨率。在小鼠，大鼠和鸣禽脑组织，但该协议已经得到验证，预计将很容易适应其他脊椎动物物种，以及非神经组织的数组。在这部影片中，我们提供详细演示了此过程的主要步骤。
Protocol
该协议是基于标准的放射性和非放射性原位杂交方法，以前我们和其他人开发的检测在脑组织1-7中的一个或两个成绩单物种的发展和完善。下面所描述的协议有2或3天的总长度，这取决于由最终用户选择的程序中断的数量。下面详细的所有步骤都riboprobe杂交步骤和杂交后洗外，在室温下进行。在此方法中涉及的所有步骤所需的解决方案和缓冲区，可以发现在此协议结束。 <p class="…
Discussion
我们使用这个协议来研究脊椎动物的大脑是如何neurochemically和功能组织，并确定如何行为相关的感官刺激的影响在成人大脑 8-10元基因组机械。我们已经成功地使用这种方法在小鼠，大鼠和鸣禽脑组织，但预计此协议将很容易适应从脊椎动物物种的阵列，或许非神经组织得到的脑切片。需要获得可靠的结果和最佳的信号的关键控制已经详细广泛，本集团其他地方11，12 。
Disclosures
The authors have nothing to disclose.
Acknowledgements
NIH / NIDCD和施密特基金会到RP的赠款支持的工作。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| DEPC | VWR | IC15090225 | toxic substance | |
| PCR purification kit | QIAgen | 28104 | ||
| Formaldehyde | VWR | BDH0506-4LP | toxic substance | |
| T3 RNA polymerase | Roche | 11031163001 | ||
| T7 RNA polymerase | Roche | 10881767001 | ||
| Tris-HCl (1 M, pH 7.5) | VWR | IC816124 | ||
| MgCl2 (1 M) | Sigma | 449172 | ||
| Spermidine (1 M) | Sigma | S0266 | ||
| DIG RNA labeling mix | Roche | NC9380805 | ||
| Biotin RNA labeling mix | Roche | NC9440104 | ||
| RNasin | Promega | N2111 | ||
| BSA | Sigma | 05491 | ||
| DTT | Sigma | D5545 | ||
| Sephadex G50 | VWR | 95016-772 | ||
| EDTA | VWR | 101384-758 | ||
| SDS | Sigma | L4390 | ||
| Transfer (t)RNA | Invitrogen | 15401011 | ||
| 10X MOPS | VWR | 14221-398 | toxic substance | |
| Paraformaldehyde | VWR | AAAL04737-36 | toxic substance | |
| Sodium phosphate monobasic | Sigma | S3139 | ||
| Sodium phosphate dibasic | Sigma | S3264 | ||
| NaOH | VWR | SX0600-1 | ||
| Triethanolamine | VWR | IC15216391 | ||
| Acetic anhydride | VWR | MK242002 | toxic substance | |
| SSPE | VWR | 82021-488 | ||
| Formamide | VWR | JT4028-1 | toxic substance | |
| Poly A | Invitrogen | POLYA.GF | ||
| Mineral oil | VWR | IC15169491 | ||
| Chloroform | VWR | BDH1109 | organic solvent | |
| Deionized formamide | Sigma | F9037 | toxic substance | |
| Hydrogen peroxide | VWR | VW36901 | toxic substance | |
| Tween 20 | Sigma | P1379 | ||
| Triton X-100 | J. T. Baker | X198-05 | ||
| Anti-DIG HRP | Roche | 11093274910 | ||
| Anti-biotin peroxidase | Vector | SP3010 | ||
| TSA Alexa Fluor 594 | Invitrogen | T20935 | ||
| TSA Alexa Fluor 488 | Invitrogen | T20932 | ||
| Hoechst | VWR | 200025-538 | ||
| Vectashield | Vector | H-1000 | ||
| embedding mold | VWR | 15160-157 | ||
| cryostat | Leica | CM 1850 | ||
| Superfrost plus slide | VWR | 89033-052 |
Solutions
- Column washing buffer: 10 mM Tris-HCl, 0.15 M NaCl, 0.05 mM EDTA, 50 μg/μl tRNA, 0.1% SDS in 50 ml DEPC-treated water.
- Column blocking buffer: 10 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA in 50 ml DEPC-treated water.
- TNT buffer: 60 ml of 1 M Tris-HCl, 18 ml of 5 M NaCl and 1.8 ml of Triton X-100 in 600 ml of DEPC-treated water.
- TNB buffer: 100 mM Tris-HCl, 8.3 μg/μl BSA, 0.15 M NaCl and 3% Triton X-100 in DEPC-treated water.
- TE buffer: 10 mM Tris-HCl, pH 7.5 plus 1 mM EDTA, pH 7.5.
- Acetylation solution: 2.7 ml of triethanolamine plus 0.5 ml of acetic anhydride in 200 ml of DEPC-treated water.
- Hybridization solution: 50% formamide, 2X SSPE, 2 μg/μl tRNA, 1 μg/μl BSA and 1 μg/μl poly A in DEPC-treated water. It needs 16 l per section.
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Cite This Article
Jeong, J. K., Chen, Z., Tremere, L. A., Pinaud, R. Double Fluorescence in situ Hybridization in Fresh Brain Sections. J. Vis. Exp. (42), e2102, doi:10.3791/2102 (2010).