Cerebellum Dissection and Slice Preparation: A Method to Generate Tissue Slices from Mouse Cerebellum

Abstract

Source: Thetiot, M. et al. Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures. J. Vis. Exp. (2019)

This video describes a method to generate tissue slices from the mouse cerebellum. The cerebellar slices can be used for organotypic culturing that provides insights into neurodevelopmental mechanisms, including myelination dynamics.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Cerebellum Dissection and Sections Preparation (Hands-on Time ≈ 15–20 min per animal) Place one of the 60 mm cell culture dishes containing the ice-cold dissection medium under the binocular microscope and remove the top plate. Swab the head fur with 70% ethanol (carefully avoiding …

Representative Results

Figure 1: Illustration of cerebellum slices generation. The dissection is divided in three main steps. (A) The cerebellum is isolated, and the meninges removed (steps 1.10 and 1.11). (B) The cerebellum is then transferred to the chopper platform and sliced (steps 1.12 to 1.16). (C) Lastly, the slices are isolated from the vermis …

Materials

BME medium	ThermoFisher Scientific	41010026
Hank’s Balanced Salt Solution (10x HBSS)	ThermoFisher Scientific	14180046
GlutaMAX (100x)	ThermoFisher Scientific	35050038
Heat-inactivated Horse Serum	ThermoFisher Scientific	26050088
Penicillin–Streptomycin (10.000 IU/mL)	ThermoFisher Scientific	15140122
Gey’s Balanced Salt Solution	Sigma Aldrich	G9779-500ML
D-Glucose Solution (45%)	Sigma Aldrich	G8769-100ML
Paraformaldehyde (PFA)	Electron Microscopy Sciences	15714
Absolute ethanol (100% ethanol)	VWR Chemicals	20821.33
Triton® X-100	Sigma Aldrich	X100-500ML	Cooled at -20°C
10% Normal Goat Serum (NGS)	ThermoFisher Scientific	500622
Phosphate Buffer Solution	EuroMEDEX	ET330-A
Tissue chopper	McIlwain		pH 7.4
Razor blades
Large scissors	F.S.T	14101-14
Small scissors	F.S.T	91500-09
Fine-straight forceps	F.S.T	91150-20
Curved-fine forceps	F.S.T	11297-00
Cell culture dishes (60 mm and 100 mm)	TPP
4-, 6-well culture plates	TPP
Millicell culture inserts (0,4 µm, 30 mm diameter)	Merck Millipore	PICM0RG50
Cell culture incubator
Fine-end pipette tips	Dutscher	134000CL	37°C, 5% CO2
Wide-bore pipette tips	ThermoFisher Scientific	2079G
Sterile syringe	Terumo Europe
Sterile syringe filters	Terumo Europe		20 or 50 mL
Scalpel	Swann-Morton	510	0.22 µm
Brush
Microscope slides	RS France
Glass coverslips	RS France		76 mm x 26 mm x 1.1 mm
Kimtech Sciences Tissue Wipers	Kimberly-Clark Professional	5511	22 mm x 22 mm
Binocular microscope