Jul 15, 2022

Public workspaceCryosectioning mouse brain

DOI
dx.doi.org/10.17504/protocols.io.e6nvw54jdvmk/v1
  • 1Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA, Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase MD 20815 USA;
  • 2Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA, Department of Neurology Longhua Hospital Shanghai University of Traditional Chinese Medicine Shanghai 200032 China
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DOI: dx.doi.org/10.17504/protocols.io.e6nvw54jdvmk/v1
Protocol Citation: Pranay Srivastava, Waijiao Cai, Xiqun Chen 2022. Cryosectioning mouse brain. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw54jdvmk/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: June 16, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 50814
Keywords: Cryosectioning, Mouse brain, ASAPCRN
Abstract
This protocol details the cryosectioning of mouse brain.
Attachments
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ds8vbjtc7.pdf
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Cryosectioning mouse brain
Cryosectioning mouse brain
1w 0d 0h 0m 20s
1w 0d 0h 0m 20s
Place the mouse brain in the mouse brain slicer (TemperatureOn ice ). Cut the hinder of the brain and put it in 4% PFA DurationOvernight at Temperature4 °C .

Overnight
Change the solution to 30% sucrose/PBS at Temperature4 °C until the brain sinks in the bottom (DurationOvernight ).

Overnight
Change the solution to 60% sucrose/PBS at Temperature4 °C for Duration48:00:00 -Duration120:00:00 (optional).

1w
Cut 1/2 to 2/3 of the cerebellum to make a flat seat.
Cut the pieces of foil for the tissue wrapping and label the pieces with chemical resistant marker.
Place a small beaker containing 2/3 2-methylbutane, at least half buried in dry ice. Allow it to cool enough (when you see it turn white and thick at the bottom of the container).
Gently place the mouse brain into 2-methylbutane and allow it to sit for a while until it no longer “smoking” (~Duration00:00:20 ).

20s
Place a layer of OCT on the foil on dry ice. Wait a few seconds until OCT turns to half opaque.
Place the brain on the layer of OCT and place another layer of OCT to cover the brain.
Wrap the brain with the labeled foil and store the brain at Temperature-80 °C until sectioning.

Adjust the cryostat to: chamber Temperature-22 °C , specimenTemperature-23 °C , Amount30 μm .

Place the brains in cryostat when it reaches Temperature-22 °C for half hour to allow the temperature balanced.

In 12-wells plate, add Amount1.5 mL cryo-protective solution (30% Glycerol, 30% Ethylene Glycol in PBS) per well.

Pipetting
For stereological dopaminergic neurons counting, start to collect the sections when it close to substantial nigral (midbrain is apple-ish like shape. Hippocampus spread to 2/3 of the cortex). Collect ~10-12 sections per well. Store plates at Temperature-20 °C until staining.
Note
ABCDE
1234
A Mouse# 1Section 1,5,9,13,17,21,25, 29Section 2,6,10,14,18, 22, 24, 30Section 3,7,11,15,19,23,25, 31Section 4,8,12,16,20,24,28, 32
B Mouse# 2
C Mouse# 3