Sample preparation

The following are hints for preparing samples

Perfusion

• Perfuse as normal in 4% PFA. Do not drop-fix.

• Take great care removing the brain from the skull. Aim for zero damage. Use a dissection scope if it helps. Keep the olfactory bulbs and the cerebellum.

• If you see dura adhering to the surface, remove it with care.

• Post-fix overnight in 4% PFA at 4 degrees C. This is more important for rat brains: these may need two days.

• Transfer to 1x PBS.

• Sucrose is not involved: we aren't using a cryostat.

• Longer term storage (months) is not encouraged because brains deform over time. If necessary, however, use 0.01% azide and label as such. Do not use higher azide concentration: that stuff is really dangerous.

Imaging solution

• Use PB not PBS for the water bath solution. PBS seems to cause corrosion of components. 50 mM PB is adequate: we just need to buffer pH.

• Brains can be stored in 1x PBS before being imaged. Brains coming straight from PFA should be equilibrated for at least 12 hours in 50 mM PB before being imaged.

• Do not store imaging PB in the fridge: you will get air bubbles as it warms.

PB Protocol

To make 2L of 50 mM PB:

• Add 3.1 g of sodium phosphate monobasic monohydrate (S9638-1KG) to 2L of water. Stir and dissolve.

• Once the above is dissolved add 20.8 g of sodium phosphate dibasic heptahydrate (CAS: 7782-85-6). Do add this until the first component has dissolved or you will get a precipate.