为揭示复杂的大脑地形wholemount免疫组化

Summary

神经回路地形组织功能的车厢与特定的分子型材。在这里，我们提供一个多功能wholemount免疫组织化学染色方法为揭示全球脑地形使用的实际和技术步骤。我们证明了实用的使用方法很好理解的小脑细胞构筑和电路。

Abstract

反复和充分了解小脑的细胞结构，使其理想的模型系统，为探索脑地形。其相对统一的细胞结构的基础是一系列复杂的基因和蛋白表达的矢状域。小脑分子条块分割镜像传入纤维的解剖和功能的组织。要充分认识小脑组织的复杂性，我们以前完善了wholemount染色方法在小鼠小脑的图案缺陷的高通量分析。该协议详细描述的试剂，工具，是有用的实际步骤，成功地揭示了成年小鼠小脑使用wholemount染色蛋白表达模式。这里展示的步骤强调使用这种方法的效用作为一个例子，如何可以在其透露的大脑的精细地形表达zebrinII / aldolaseC的原生立体构象。还介绍了协议允许传入的预测和大量分子地形比较研究小脑蛋白表达的可视化的适应。为了说明这些应用程序，从大鼠小脑的传入染色的数据。

Protocol

1。动物灌注和小脑剖析根据蛋白质，灌注可能是必不可少的成功染色1,2。 transcardiac灌注是一种侵入性，非生存的过程，需要正确使用的麻醉药。正确的培训，机构批准，并IACUC批准，都在尝试的过程之前，必要的。它始终是一个好主意，咨询机构的兽医，以确定实验的要求，并获得正确的训练中得到的帮助。手术前，动物应该是深度麻醉，没有反应，如脚或尾巴捏刺激。用剪刀?…

Discussion

我们已经介绍了使用一个多功能的免疫组织化学方法，在发展中国家和成人的大脑暴露蛋白表达为成功wholemount的染色所需的技术细节。复杂的分子的表达方式，通过使用这种方法，可以分析和脑地形而不需要费时费力组织切片程序赞赏。

该协议已被用于揭示成人^1,2,8,9和产后早期小鼠小脑^10,11,12图案几个Purkinje细胞蛋白表达。我们原来的研究也表明了实用的的?…

Materials

Materials	Function in protocol
Perfusion pump (Fisher Scientific/13-876-2)	Allows for consistent and slow perfusion.
Sharp-tip Scissors (FST/14081-08)	General use in perfusion and dissection.
Blunt-tip Forceps (FST/91100-12)	To stabilize the heart for insertion of the perfusion needle.
Forceps (FST by Dumont AA/11210-10)	For use during dissection of the brain from the skull and to separate the cerebellum from the rest of the brain. These are essential because they have a slightly rounded tip that helps minimize damage to the cerebellum during dissection.
Nutator (Fisher Scientific)	Used to keep tissue in motion during incubation periods.
1.5 mL tube (Sarstedt/Screw Cap Micro Tube)	All steps of the histochemistry protocol take place in these microtubes. The rounded bottom ensures that the cerebellum stays in motion.
Perforated spoon (FST/10370-17)	Used to keep wholemounts in the microtubes while gently decanting out the spent solution.
Leica MZ16 FA microscope	Used to examine wholemount staining.
Leica DFC3000 FX camera	Used to capture wholemount images.

Table 1.

Example calendar for a typical wholemount experiment
Day 1	Dent’s fix, room temperature, 8 hrs				Dent’s bleach, 4°C, overnight
Day 2	100% MeOH, room temperature, 2x, 30 min each			100% MeOH, Freeze/thaw, 4x, 30 min/15 min			100% MeOH, -80°C, overnight
Day 3	50% MeOH/50% PBS, room temperature, 60-90 min	15% MeOH/ 85% PBS, room temperature, 60-90 min		100% PBS, room temperature, 60-90 min		10μg/mL Proteinase K in PBS, room temperature, 2-3 min	100% PBS, room temperature, 3x, 10 min each	PMT, 4°C, overnight
Day 4-5	PMT + 1° antibody + 5% DMSO, 4°C, 48 hrs
Day 6	PMT, 4°C, 2-3x, 2-3 hrs each			PMT + 2° antibody + 5% DMSO, 4°C, 24 hours (Or begin amplification steps with ABC complex)
Day 7	PMT, 4°C, 2-3x, 2-3 hrs each		PBT, room temperature, 2 hrs			Incubate in fresh DAB in PBS until optimal staining is visualized

Table 2.

Recipes (*=prepare fresh every time)
PBS (phosphate buffered saline)	0.1M phosphate buffered saline in deionized water. pH 7.2 (Sigma tablets; P4417)
PFA (Paraformaldehyde)	Made and stored frozen as a 20% solution and then diluted to 4% in PBS for the working solution (Fisher Scientific; T353)
Dent’s Fixative3*	4 parts methanol 1 part dimethylsulfoxide (DMSO; Fisher Scientific; D159-4)
Dent’s Bleach3*	4 parts methanol 1 part dimethylsulfoxide (DMSO; Fisher Scientific; D159-4) 1 part 30% hydrogen peroxide
Enzymatic Digestion	10 μg/ml of Proteinase K (Roche Diagnostics; 03115828001) in PBS.
PBST	PBS containing: 0.1% Tween-20 (Fisher Scientific, BP337; Triton can also be used in place of Tween-20 in all instances.)
PMT25*	PBS containing: 2% nonfat skim milk powder (Carnation preferred) 0.1% Tween-20 (Fisher Scientific; BP337)
PBT25*	PBS containing: 0.2% bovine serum albumin (Sigma; B9001S) 0.1% Tween-20 (Fisher Scientific; BP337)
DAB*	Dissolve one 10-mg tablet of 3,3-diaminobenzidine (Sigma-Aldrich; D5905) in 40 ml of PBS. Add 10 μl of 30% hydrogen peroxide to initiate reaction).
ABC Complex Solution	Vectastain kit (Vector laboratories, Inc; PK-4000)

Table 3.