RE: Mouse brain cryostat sections

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From:Emma Carter <> (by way of histonet)
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> On Fri, 17 Dec 1999, David Sanan wrote:
> > This isn't long enough, though you may need minimal fixation.
>  > It depends what you want to do with the sections.
> (EC) I always used to fix my brains overnight in the para at 4'....but
> these were whole mounts.
>    >  You have not done a cryoprotection. (OCT is just physical
>    > support around the outside of the specimen. You need to
>    > soak the fixed brain in sucrose (15 to 30%; the stronger
>    > the better) until it sinks (e.g. 24 h at 4C).
> (EC) I have always used 30% sucrose in PBS, left at 4' until the
> brains sank..this could sometimes take as long as over the weekend
> (once they have been taken out of the para, there is no risk of
> further and consequently over-  fixation).
> I would then freeze the brains in dry ice.
>     >  That's not true at all. It takes about 10 minutes to
>     > perfuse an anaesthetized mouse with 5-10 ml of saline
>     >followed by about 25 ml of buffered formaldehyde, and
>     >the capillaries certainly don't clog! This is the fastest
>     >and best way to deliver fixative to cells in the brain.
>     >Many research workers say it's the only acceptable way.
> (EC) When  i did perfusions, i would flush through with a
> heparin-saline solution for about 5 minutes, followed by para
> perfusion for a fair time, up to 10 minutes.....(without being
> graphic, the body stiffens as the para gets to work....), then dissect
> out the brain...
> ...then put it in para overnight, before transferring to a sucrose/PBS
> solution till it sinks........then freeze on dry ice (in OCT if
> required)
> Hope this helps!!!
> Happy has snowed here in oxfordshire, and the roads
> are hardly ever snows the week before
> la la!!
> emma carter
> Oxford BioMedica (UK) Ltd

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