Sample preparation
The following are hints for preparing samples


    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.
Last modified 1mo ago