Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

Summary

We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage.

Abstract

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 μm thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 μm working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic trees and dendrite spines may be reliably visualized and quantified.

Introduction

The visualization of individual neurons within tissue samples allows for the in situ analysis of neuron morphological characteristics, which has significantly advanced our understanding of the brain and how it may be influenced by endogenous disease or exogenous environmental factors. The Golgi-Cox staining method is a cost-effective, relatively simple means of staining a random sample of neurons within the brain. First developed by Golgi¹ and modified by Cox² in the 1800s, researchers have further refined this technique over the years to produce clear, well-stained neurons that can be used to visualize and quan....

Protocol

Adult female CD1-strain mice were used in this study. Similar staining can be accomplished using both sexes at various ages. Experimental animals were cared for according to the principles and guidelines of the Canadian Council on Animal Care, and the experimental protocol was approved by the University of Guelph Animal Care Committee.

1. Golgi-Cox Staining

1. Golgi-Cox Impregnation of Brains
   1. Make the Golgi-Cox solution of 1% (w/v) potassium dichromate, 0.8% (w/v) potassium chromate and 1% (w/v) mercuric chloride by dissolving the potassium dichromate and potassium chromate into high-quality water separ....

Results

This Golgi-Cox staining protocol and its two described optional variants may be employed to visualize individual neurons within 400 - 500 μm thick brain sections. Representative image montages of two-dimensional Z-projections captured using a 10X objective and 5 μm steps in the Z axis are shown in Figure 1: A1 - C1 for a large area of coronal brain sections that includes the anterior cingulate cortex area 1 and the secondary motor cortex¹⁸. Section.......

Discussion

We describe here a Golgi-Cox staining protocol along with two useful variants for visualizing neurons within thick brain sections. As shown in the Representative Results, the use of a high-resolution objective that has a long 800 μm working distance allows for the reliable visualization of entire neurons throughout the depth of brain sections cut at 500 μm. This study of relatively thick brain sections increases the probability that stained neurons of any type are fully contained within the slice, which is espe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
potassium dichromate	Fisher Scientific	P188-100	Hazardous
potassium chromate	Fisher Scientific	P220-100	Hazardous
mercuric chloride	Fisher Scientific	S25423	Hazardous
Whatman grade 1 filter paper	Fisher Scientific	1001-185
isoflurane	Pharmaceutical Partners of Canada	CP0406V2
20 mL scintillation vial	Fisher Scientific	03-337-4
sucrose	Bioshop Canada	SUC700.1
sodium phosphate monobasic	Sigma Aldrich	S5011-500G
sodium phosphate dibasic	Sigma Aldrich	S9390-500G
50 mL conical tube	Fisher Scientific	12-565-271
isopentane	Fisher Scientific	AC126470010	Also known as 2-methylbutane; hazardous
agar	Sigma Aldrich	A1296-100G
small weigh dish	Fisher Scientific	02-202-100
vibratome	Leica	VT1000 S
6-well tissue culture plates	Fisher Scientific	08-772-1b
mesh bottom tissue culture inserts	Fisher Scientific	07-200-214
paraformadelhyde (PFA), 16%	Electron Microscope Sciences	15710-S	Hazardous
ammonium hydroxide	Fisher Scientific	A669S-500	Hazardous
Kodak Fixative A	Sigma Aldrich	P7542
superfrost plus slides	Fisher Scientific	12-550-15
CitroSolv clearing agent	Fisher Scientific	22-143-975
anhydrous ethyl alcohol	Commercial Alcohols	N/A
cresyl violet	Sigma Aldrich	C1791
permount	Fisher Scientific	SP15-100
upright microscope	Olympus	BX53 model
colour camera, 12 bit	MBF Biosciences	DV-47d	QImaging part 01-MBF-2000R-F-CLR-12
3D motorized microscope stage, controller and enoders	MBF Biosciences	N/A	Supplied and integrated with microscope by MBF Biosciences
4X microscope objective	Olympus	4x 0.16 N.A. UplanSApo
10X microscope objective	Olympus	10x 0.3 N.A. UPlan FL N
30X microscope objective	Olympus	30x 1.05 N.A. UPlanSApo
60X microscope objective	Olympus	60x 1.42 N.A. PlanAPO N
silicone immersion oil	Olympus	Z-81114
Neurolucida software	MBF Biosciences	Version 10
ImageJ software	U. S. National Institutes of Health	Current version	With the OME Bio-Formats plugin installed
Photoshop software	Adobe	version CS6