Equipment :
Eppendorf EDOS 5222 Liquid dipensing system
CamLab Omni-Mixer ES Homogeniser
Class 2 Microbiological Safety Cabinet

Solution :
0.25M-sucrose (MW 342.30) : 85.56g/L in pyrogen-free water.
Pre-chilled to ~4 degrees centigrade

Procedure:

1. Weigh frozen brain regions. Thaw to allow slicing into 4-5g chunks and transfer to homogenisation container.
2. Homogenise to yield 10% (w/v) homogenate solution in sealed 250 mL container using programmable Omni-Mixer. Note due to weight of obex a 5% (w/v) homogenate was prepared. Setting at 4000rpm, 6 x 30seconds.*
3. Place homogenate bottle on ice until ready for dispensing.
4. Wash homogenisation bit by running machine (washing step in done between homogenisation of different regions of the brain); Setting at 4000rpm, 6 x 30seconds with sucrose solution.
5. Dispense 100 x 0.5 mL into pre-labelled, Simport 1.2mL cryovials, immerse in bleach (10%-Chloros) for >15 minutes at 4^oC.
6. Rinse in water to remove surface bleach. "Flash Freeze " by immersing in liquid nitrogen for 10 minutes.
7. Transfer onto dry ice for transport to storage facility at NIBSC (-80 ^oC).

* Shown to be adequate by facsimile homogenisation of frozen sheep brain