Cerebellum Dissection and Slice Preparation: A Method to Generate Tissue Slices from Mouse Cerebellum
Published: April 30, 2023
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 …
Açıklamalar
The authors have nothing to disclose.
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 |
Etiketler
Bu Makaleden Alıntı Yapın
Cerebellum Dissection and Slice Preparation: A Method to Generate Tissue Slices from Mouse Cerebellum. J. Vis. Exp. (Pending Publication), e20696, doi: (2023).