TITLE: mouse brain storage and sectioning 
AUTHORS: Tony Hsiao

[DESCRIPTION]
detailed protocol to receive mouse brain tissue from US to Australia including storage and sectioning 

[STEPS]
SECTION: Storage and sectioning of mouse brain tissue
1. Archive mice brains for storage 
SECTION: Storage and sectioning of mouse brain tissue
1.1. Sample arrive in the delivery room (4°C)
SECTION: Storage and sectioning of mouse brain tissue
1.2. Move samples from the delivery room to PC2 laboratory. 
SECTION: Storage and sectioning of mouse brain tissue
1.3. Cross check tissue ID with shipping log
SECTION: Storage and sectioning of mouse brain tissue
1.4. Inspect for floating tissue in 30% sucrose and discoloration.
SECTION: Storage and sectioning of mouse brain tissue
1.5. Inspect for floating tissue in 30% sucrose and discoloration.
SECTION: Storage and sectioning of mouse brain tissue
1.6. Prepare labelled (date of arrival and tissue ID) 50ml falcon tubes filled with new sucrose solution (30% sucrose, 0.02% sodium azide).
SECTION: Storage and sectioning of mouse brain tissue
1.7. Pour old sucrose out and gently place tissue into the new falcon tubes.
SECTION: Storage and sectioning of mouse brain tissue
1.8. Store tubes in 4°C on shaker overnigh.
SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
2. 30% sucrose solution

SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
2.1. Dissolve 300g of sucrose in 300mL of distilled water
SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
2.2. Add 100mL of 10x PBS
SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
2.3. Adjust final volume to 1L with distilled water.

SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
2.4. Add 2mL of 10% sodium azide to solution.

SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
2.5.  Mix well until solution is homogenous and store in 4°C
SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
3. 1X Phosphate Buffer (1M)
SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
3.1. In 1.8L of dH2O, add 38.7g of NaH2PO4.H2Oand 101.9g of Na2HPO4.
SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
3.2. Mix solution until it is homogenous and store in 4°C.


SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
4. Cryoprotectant Solution for Mouse Brain (1L)
SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
4.1. Add 300mL of glycerol, 300mL of ethylene glycol, 100mL 1xPhosphate
Buffer, and 300mL of dH2O

SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
4.2.  Mix solution overnight on the stirrer at RT. 

SECTION: Sucrose solution/PB buffer/Cryoprotectant solution for mouse brain (1L)
4.3.    
Store solution at 4°C
SECTION: tissue cutting and storage
5. Mice brain sectioning and storage 
SECTION: tissue cutting and storage
5.1. 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. 
SECTION: tissue cutting and storage
5.2. Create a rectangle mould using metal plates to fit the mouse brain.
SECTION: tissue cutting and storage
5.3. Pour Clear O.C.T Compound Embedding Medium to the mould up to ¼ of the volume. Ensure that the medium is evenly distributed. 
SECTION: tissue cutting and storage
5.4. Place the mould filled with embedding medium inside the Epredia‱
CryoStar‱ NX50 chamber and use the quick freeze option to quickly freeze the
medium
SECTION: tissue cutting and storage
5.5. Move sample from the cool room to RT
SECTION: tissue cutting and storage
5.6. Retrieve the mould with frozen embedding medium and pour more medium up
to ½ of the volume
SECTION: tissue cutting and storage
5.7. Gently place the mouse brain to the mould using forceps, submerging it
in embedding medium. Adjust the orientation as needed
SECTION: tissue cutting and storage
5.8. Slowly pour more embedding medium on top of the mouse brain until it is
completely submerged in embedding medium
SECTION: tissue cutting and storage
5.9. Place the mould back inside the Epredia‱ CryoStar‱ NX50 chamber and
quickly freeze it
SECTION: tissue cutting and storage
6. 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
SECTION: tissue cutting and storage
6.1. Pour a 1cm diameter dollop on the circular cutting plate
SECTION: tissue cutting and storage
6.2. Release the mould and place the frozen tissue on the cutting plate,
front orientation up
SECTION: tissue cutting and storage
6.3. Place the tissue and cutting plate into the chamber, quickly freezing it
SECTION: tissue cutting and storage
6.4. Place the frozen tissue and cutting plate to the cutting table. Adjust
the orientation so that the mould is parallel to the cutting blade.

SECTION: tissue cutting and storage
6.5. Cut PF with 20um of thickness and place it in the PF vial. 
SECTION: tissue cutting and storage
6.6. Cut ST with 20um of thickness and sequentially place them in 8 vials,
repeat the process until SN is reached

SECTION: tissue cutting and storage
6.7. Change section thickness to 30um to cut SN and sequentially place them
in 8 vials, repeat the process until the cerebellum is reached.
SECTION: tissue cutting and storage
6.8. Release the frozen cerebellum and cutting plate from the cutting table

SECTION: tissue cutting and storage
6.9. Remove the frozen cerebellum from the cutting plate and place it into the vial for
leftover. 


SECTION: tissue cutting and storage
6.10. Vials are then placed into designated boxes and stored in -20°C