Quantifying Newly Generated Neurons in Rat Brain Tissue Using the Optical Fractionator Technique

Abstract

Source: Olesen, M. V., et al. The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy. J. Vis. Exp. (2017).

The video demonstrates the technique for quantifying newly generated neurons in brain tissue. Hippocampal tissue slices containing Bromodeoxyuridine (BrdU)-labeled neurons were stained using immunohistochemistry and subsequently quantified across the tissue thickness using the optical fractionator technique.

Protocol

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

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5'-bromo-2'deoxyuridine - BrdU	Sigma-Aldrich	19-160
Cresyl Violet	Sigma-Aldrich	C5042	Contains the following materials for 1 L: 0.2 g cresyl violet; 2.0 g sodiumacetate, 3 H2O; 3 ml glacial acetic acid; 1000 ml destilled water
Cryoprotectant	Capital Region Pharmacy, DK	861334	Contains the following materials: Sucrose; polyvinylpyrrolidon PVP 40; sodiumphosphatebuffer (0.2 M); ethylenglycol; demineralized water.
dH2O	Capital Region Pharmacy, DK	817205
Diaminobenzidine – DAB	Sigma-Aldrich	D5637
Envision-HRP	DAKO	K4001	Peroxidase labeled polymer conjugated to goat anti-mouse immunoglobulins
Ethanol - 70 %	Plum A/S	201098
Ethanol - 96 %	Plum A/S	201104
Ethanol - 99.9 %	Plum A/S	201111
Fetal Bovine Serum	In Vitro	BI-04-003-1A
H2O2 30%	Capital Region Pharmacy, DK	896456
HCl	J. T. Baker	6081
Mounting medium	Sakura	4583	Tissue Tek
Mouse-anti-BrdU	Becton-Dickinson	347580
PBS buffer	Capital Region Pharmacy, DK	853436	pH = 7.4
PFA	VWR	28,794,295	pH = 7.4
Phosphate buffer	Capital Region Pharmacy, DK	866533	0.1 mol/l, pH 7.4
Rapid drying mounting medium	Histolab Products AB	801	Pertex
Sodium Tetraborate	Merck A/S	1,063,080,500
Sucrose	Merck A/S	1,076,515,000
Triton X-100	Merck A/S	1,086,031,000
Xylene	VWR	28,973,363
Cover slips	Menzel-Gläser	24x50 mm #0
Cryostat	Leica	CM1860
Digital Microcator	Heidenhain	VRZ 401
Glass dishes	Brain Resaerch Laboratories	1256
Microscope	Nikon	Eclipse 80i
Microscope camera	Olympus	DP72
Microscope slides	VWR	48311-703	SuperFrost+
Motorized stage	Prior	H101A
Multidish Containers	Sigma-Aldrich	D6315-1CS	Nuclon 12-wells
New-Cast software	Visiopharm	ver. 4.5.6.440
Objective 2x	Nikon	CFI Plan UW 2X
Objective 4x	Nikon	CFI Plan Fluor 4X
Objective 10x	Nikon	CFI Plan Fluor 10X
Objective 100x oil immersion	Nikon	CFI Plan Apo Lambda DM 100X Oil	Numerical aperture 1.40
Orbital shaker	Gemini BV	CM-9	Sarstedt
Slide rack	Sakura	4465
Slide staining dishes	Sakura	4457	Tissue-Tek
Specimen disc	Leica	14037008637
Staining nets	Brain Research Laboratories	2512	25-wells