Wholemount Immunohistochemistry for Revealing Complex Brain Topography

Summary

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Abstract

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Protocol

1. Animal Perfusion and Cerebellum Dissection Depending on the protein, perfusion may be essential for successful staining1,2. Transcardiac perfusion is an invasive, non-survival procedure that requires the proper use of anesthetics. Correct training, institutional approval, and IACUC approval are all necessary before attempting the procedure. It is always a good idea to consult the institution’s veterinarians to get help in identifying experimental requirements and acquiring the correct training. …

Discussion

We have described the technical details required for successful wholemount staining using a versatile immunohistochemical approach for revealing protein expression in the developing and adult brain. By using this approach, complex molecular expression patterns can be analyzed and brain topography appreciated without the need for laborious and time consuming tissue sectioning procedures.

This protocol has been used to reveal the patterned expression of several Purkinje cell proteins in both the…

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.