Feb 16, 2025
Coronal cryosectioning of mouse brains
Forked from Coronal cryosectioning of mouse brains
- 1Caltech
Protocol Citation: Yujie Fan, Chelsie Steele 2025. Coronal cryosectioning of mouse brains. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9rkzpv3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 08, 2025
Last Modified: February 16, 2025
Protocol Integer ID: 117846
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
Cryostat coronal cryosectioning of mouse brain tissue
Materials
Leica Biosystems, CM1950cryostat (Leica Biosystems, CM1950)
Before start
PFA-perfused, 30% sucrose-cryoprotected, and flash-frozen mouse brains are stored at -80C until cryosectioning
Procedure
Procedure
25m
25m
Retrieve frozen mouse brain stored at -80 °C .
Place brain in cryostat. Set chamber temperature to -16 °C to -20 °C . Set brain holder temperature to -20 °C . Let brain equilibrate to temperature for at least 00:20:00 before sectioning.
20m
Insert chuck and blade into cryostat.
Apply a drop of OCT on the chuck, and affix brain vertically (olfactory bulbs up).
Apply another layer of OCT at the base of the brain, turning the brain-holder with the other hand. Cover the cerebellum and the junction between the cerebellum and cortex with OCT. Allow OCT to freeze solid (~00:05:00 ).
5m
Cut 100 micron sections. Adjust angle of medio-lateral and ventro-dorsal axis so that sections are free of cutting artifacts, symmetrical, and well-aligned to reference atlas.
Use of anti-roll plate is optional and can help reduce rolling of sections, but must be carefully adjusted to avoid cutting artifacts.
Collect free-floating sections in 6-well plate.
Label your plate lid and base with brain/experiment details using lab marker and/or lab tape.
Pre-fill wells with 3 mL 1X PBS containing 0.01% sodium azide (100mg/L).
Collect multiple sections per well. Typically the plate is labeled with different brain regions to differentiate each of the wells
Use parafilm to seal the lid to the plate (air-tight) and refrigerate at 4 °C until further processing.