Re: ratbrain slices 2

From:Geoff McAuliffe

Frouwke Kuijpers wrote:

> I also like to have information about fixation, processing and imbedding in
> paraffin of rat brains.

I think there are protocols for rat and mouse in the archives.

> We have a good protocol for frozen sections (40 um) and do imuno using the
> Free Floating Method.  Disadvantage of this method is that not used sections
> cannot preserve for a long time,

I have stored 20 micron sections of paraformaldehyde-fixed (only fixed 4 hours)
rat and mouse brain for several months in PBS in the refrigerator. Changed the
PBS weekly to prevent growth of moulds, etc. There is little reduction in the
immunostaining of TH, GFAP and some macrohage markers. This may not apply to the
markers you are using.

> I presume preservation of the sections will be better

Usually, but properly prepared fixed frozen rat or mouse brain is very pretty
and there is less shrinkage. Paraffin processing does cause shrinkage.

> sections can be thinner

Yes, but this is not always "better" for central nervous system. Depends on what
you are looking for.

> and less antibody can be used ($$$).

only if the sections are smaller.

> We have  paraffin-experience
> with all kind of fish and 'xenopus' (a kind of frog), the story on our
> department is that doing this on ratbrain is much more difficult but nobody
> can explain why.

If you tell us your protocol I'm sure someone could spot potential trouble

> So, is it worth trying this or is it better using  my own freezing-protocols
> and accept the disadvantages of the FF method?

Only you can decide what is best for your lab and the work it does.

> What kind of fixative (does anyone has experience with Zn-formaline, I
> already read omething about post fixation with it ?)

Freshly-prepared 4% paraformaldehyde is a good place to start. Is there anything
in the literature on the animal/antigen combo you are interested in?

> Processing schedule (Times used for a whole ratbrain)

A whole rat brain? Are you going to cut all the way through a whole rat brain?
Cutting it into pieces will make processing easier, faster and less chance of
poor processing.

> How long paraffin sections can be stored and a good immune-staining is still
> possible?

Depends on the antigen you are looking for.

> I hope somebody can make me wise,

I wish that for myself often!

> F.J.Kuijpers-Kwant
> Dept. Cellular Animal Physiology
> University of Nijmegen
> Toernooiveld 1
> 6525 ED Nijmegen
> the Netherlands

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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