Jul 15, 2024
Coronal cryosectioning of mouse brains
- 1UCSD;
- 2University of California, San Diego
Protocol Citation: Lauren Faget, Thomas Hnasko 2024. Coronal cryosectioning of mouse brains. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g713w9gwz/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: July 15, 2024
Last Modified: August 13, 2024
Protocol Integer ID: 103431
Keywords: coronal cryosectioning of mouse brains cryostat, mouse brains cryostat, coronal cryosectioning, mouse brain tissue, mouse brain, brain
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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
Before start
PFA-perfused, 30% sucrose-cryoprotected, and flash-frozen mouse brains are stored at -80C until cryosectioning
Procedure
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 16 to 30 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.
For immunohistochemistry we typically cut 30-micron sections for best results. For RNAscope we typically cut 16 to 20-micron sections.
Collect free-floating sections in 48-well plate.
Label your plate lid and base with brain/experiment details using lab marker and/or lab tape.
Pre-fill wells with 1 mL 1X PBS containing 0.01% sodium azide (100mg/L).
Can use syringe with blunt needle to collect sections after sectioning. Dip needle into PBS and collect section by gently touching it on the side of the section.
Collect 5 sections per well. Or for experiments that require finer resolution of coordinate location can collect sections serially, adding 1 section per well until the end of the plate and start back at well #A1.
Use parafilm to seal the lid to the plate (air-tight) and refrigerate at 4 °C until further processing.