Microscopic Imaging of Neurons in a Mouse Brain Section Using Golgi-Cox Metallic Staining

Abstract

Source: Louth, E. L., et al. Imaging neurons within thick brain sections using the Golgi-Cox method. J. Vis. Exp. (2017)

This video demonstrates imaging neurons in a Golgi-Cox stained thick brain section by setting upper and lower focus boundaries and capturing overlapping images. Multiple locations are imaged to ensure detailed neuronal projections, which are combined to create a 3D representation.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review 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 separ....

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, 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
Three-dimensional 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