Sample preparation

A major advantage of serial section microscopy is that it generates 3D images that are easy to register to a standard atlas and are particularly amenable to automated analyses. However, to take advantage of this the samples must be well prepared, undamaged, and well mounted. Samples that are damaged, full of blood, or mounted at extreme angles will be hard or impossible to work with later. Take sample preparation seriously!

Perfusion

Mouse Perfusion

Perfuse as normal in 4% PFA. Do not drop-fix. Do not use old PFA: opened PFA bottles can be kept for up to 3 months in the fridge.
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. Brains should sit in 1x PBS at least overnight before imaging as the higher osmolarity of 4% PFA will cause brains to expand once the enter the imaging buffer.
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.

Rat perfusion tips

For producing rat brains with all blood removed try the following:

Use pentobarbital for overdose if you are not using it already, and use enough to knock it out in around 30s. About 2-3 ml per animal.
Time between pento injection and starting perfusion should be as low as possible (under a minute). You will need a fast and precise dissection.
Perfuse with about 150 ml of PBS or PB over 2-3 minutes followed by 200 ml of PFA for 4-5 minutes. The idea is to have enough flow at a good enough speed.

The basic cause of blood is more time spent after Pento injection and before start of perfusate flow leads to internal blood clotting (more viscous with time), and becomes difficult to push out especially in the finer vessels and capillaries. As for mouse brains, rat brains should sit in 1x PBS at least overnight before imaging as the higher osmolarity of 4% PFA will cause brains to expand once the enter the imaging buffer.

Protocols

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Perfusion

Mouse Perfusion

Perfuse as normal in 4% PFA. Do not drop-fix. Do not use old PFA: opened PFA bottles can be kept for up to 3 months in the fridge.

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. Brains should sit in 1x PBS at least overnight before imaging as the higher osmolarity of 4% PFA will cause brains to expand once the enter the imaging buffer.

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.

Rat perfusion tips

For producing rat brains with all blood removed try the following:

Use pentobarbital for overdose if you are not using it already, and use enough to knock it out in around 30s. About 2-3 ml per animal.

Time between pento injection and starting perfusion should be as low as possible (under a minute). You will need a fast and precise dissection.

Perfuse with about 150 ml of PBS or PB over 2-3 minutes followed by 200 ml of PFA for 4-5 minutes. The idea is to have enough flow at a good enough speed.