Dec 18, 2023
mouse brain storage and sectioning
- Tony Hsiao1,2,
- Felicia Xaveria Suteja1,2,
- YuHong Fu1,2,
- Glenda Halliday1,2
- 1Brain and Mind Centre & Faculty of Medicine and Health School of Medical Sciences, The University of Sydney, Sydney, NSW 2050, Australia;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Tony Hsiao, Felicia Xaveria Suteja, YuHong Fu, Glenda Halliday 2023. mouse brain storage and sectioning . protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3mo5vzp/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: December 18, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 92437
Keywords: Cryostat cutting the section archive., ASAPCRN
Abstract
detailed protocol to receive mouse brain tissue from US to Australia including storage and sectioning
Storage and sectioning of mouse brain tissue
Storage and sectioning of mouse brain tissue
Archive mice brains for storage
Sample arrive in the delivery room (4°C)
Move samples from the delivery room to PC2 laboratory.
Cross check tissue ID with shipping log
Inspect for floating tissue in 30% sucrose and discoloration.
Inspect for floating tissue in 30% sucrose and discoloration.
Prepare labelled (date of arrival and tissue ID) 50ml falcon tubes filled with new sucrose solution (30% sucrose, 0.02% sodium azide).
Pour old sucrose out and gently place tissue into the new falcon tubes.
Store tubes in 4°C on shaker overnigh.
Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
30% sucrose solution
Dissolve 300g of sucrose in 300mL of distilled water
Add 100mL of 10x PBS
Adjust final volume to 1L with distilled water.
Add 2mL of 10% sodium azide to solution.
Mix well until solution is homogenous and store in 4°C
1X Phosphate Buffer (1M)
In 1.8L of dH2O, add 38.7g of NaH2PO4.H2Oand 101.9g of Na2HPO4.
Mix solution until it is homogenous and store in 4°C.
Cryoprotectant Solution for Mouse Brain (1L)
Add 300mL of glycerol, 300mL of ethylene glycol, 100mL 1xPhosphate
Buffer, and 300mL of dH2O
Mix solution overnight on the stirrer at RT.
Store solution at 4°C
tissue cutting and storage
tissue cutting and storage
Mice brain sectioning and storage
Tubes are filled with cryoprotectant to 90% of volume: 8 tubes for ST sections, 8 tubes for SN sections, 1 tube for PF sections. One tube without cryoprotectant is prepared for leftover tissues.
Create a rectangle mould using metal plates to fit the mouse brain.
Pour Clear O.C.T Compound Embedding Medium to the mould up to ¼ of the volume. Ensure that the medium is evenly distributed.
Place the mould filled with embedding medium inside the Epredia‱
CryoStar‱ NX50 chamber and use the quick freeze option to quickly freeze the
medium
Move sample from the cool room to RT
Retrieve the mould with frozen embedding medium and pour more medium up
to ½ of the volume
Gently place the mouse brain to the mould using forceps, submerging it
in embedding medium. Adjust the orientation as needed
Slowly pour more embedding medium on top of the mouse brain until it is
completely submerged in embedding medium
Place the mould back inside the Epredia‱ CryoStar‱ NX50 chamber and
quickly freeze it
Lift the frozen mould from the chamber and mark the front orientation of
the brain by drawing a line on the frozen embedding mould using a sharpie
Pour a 1cm diameter dollop on the circular cutting plate
Release the mould and place the frozen tissue on the cutting plate,
front orientation up
Place the tissue and cutting plate into the chamber, quickly freezing it
Place the frozen tissue and cutting plate to the cutting table. Adjust
the orientation so that the mould is parallel to the cutting blade.
Cut PF with 20um of thickness and place it in the PF vial.
Cut ST with 20um of thickness and sequentially place them in 8 vials,
repeat the process until SN is reached
Change section thickness to 30um to cut SN and sequentially place them
in 8 vials, repeat the process until the cerebellum is reached.
Release the frozen cerebellum and cutting plate from the cutting table
Remove the frozen cerebellum from the cutting plate and place it into the vial for
leftover.
Vials are then placed into designated boxes and stored in -20°C