Apr 05, 2024
Preparation of Free Floating Coronal Mouse Brain Sections
- 1Van Andel Research Institute
Protocol Citation: madalynn.erb Erb 2024. Preparation of Free Floating Coronal Mouse Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pzk9g2w/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 26, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94228
Keywords: ASAPCRN
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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
Protocol to prepare free floating mouse brain sections for immunostaining or in situ hybridization.
Collect Mouse Brain Tissue
Collect Mouse Brain Tissue
Deeply anesthetize mice via intraperitoneal injection of 2X Avertin solution
Perform transcardial perfusion using chilled saline solution
0.9% NaCl kept on ice
Use approximately 60mL saline solution per mouse
Switch from saline solution to chilled PFA
4% paraformaldehyde in 0.1M phosphate buffer (PB) pH 7.4
Use approximately 60mL PFA per mouse
Remove brain immediately after PFA perfusion
Incubate brain in PFA for 24 hours at 4°C
Transfer brains to 30% Sucrose / 0.1M Phosphate Buffer (PB) – keep at 4°C for ≥ 24 hours
Tissue should be completely saturated with sucrose before sectioning
Brains will sink to the bottom of the vial when saturated
Section Tissue
Section Tissue
Use a Leica SM2010R Microtome
Blade: Leica 16cm, knife angle set at 0 degree
Cut thickness: 35µm
Adjust the microtome platform so that it is level with the blade and lock it into place
Chill the microtome platform by covering it with crushed dry ice
Apply 5-10 drops of 30% Sucrose / 0.1M Phosphate Buffer (PB) solution to the chilled microtome platform and wait for it to solidify
Use the microtome to gently shave the solidified sucrose to make a flat surface
Use a razor blade to remove any spinal cord from the brain making a flat surface perpendicular to the rostral / caudal axis
Apply 2-3 drops of sucrose to the existing sucrose platform and quickly position the brain with the olfactory bulbs pointing upwards
Apply 2-3 more drops of sucrose to the top of the brain to securely freeze it to the microtome platform
Gently cover the brain in crushed dry ice to freeze the tissue
Adjust the microtome platform so that the rostral / caudal axis is perpendicular to the blade and the dorsal / ventral axis is level with the blade
Collect sections in a 24-well plate prefilled with cryoprotectant solution (0.1M PB +30% Sucrose + 30% Ethylene Glycol)
Seal plate with parafilm and store tissue stored at -20°C
Recipes
Recipes
0.2M Phosphate Buffer (PB)
| A | B | |
| 1L | H2O | |
| 22.72 g | Na2HPO4 | |
| 5.52 g | NaH2PO4 |
4% PFA
| A | B | |
| 500mL | 8% paraformaldehyde | |
| 500mL | 0.2M PB |
Saline
| A | B | |
| 1L | H2O | |
| 9 g | NaCl |
30% Sucrose
| 300g | Sucrose | |
| Fill to 1L | 0.1M PB |
- Stir and heat to 60°C until dissolved
Cryoprotectant Solution
| A | B | |
| 300g | Sucrose | |
| 300mL | Ethylene glycol | |
| Fill to 1L | 0.1M PB |
Completely dissolve sucrose in 0.1M PB before adding ethylene glycol. After adding ethylene glycol, fill to 1L with 0.1M PB.