Re: IHC to see degenerative axon fibers
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | Maria Mejia <maria@mail.ski.org> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
On Tue, 18 Jul 2000, Maria Mejia wrote:
> Thank you so very much for the reply. I went through my books and
> finally found the protocol for this silver -Nauta-Gygax. My good-
> ness the fixating time 1 week or longer (Nauta and Gygax re-
> commend 1-3 months)!!! I know it says this in the protocol, but
> in your experience what would be a good fixating time?? We were
> hoping to perfuse w/4% PFA/0.1M phosphate buffer, then remove
> the brain and fix for an additional week???
This will probably be OK. Don't fix for less than one
week. Use the formalin recommended in N & G's original
method, which probably isn't buffered (can't remember).
> ... I haven't done a
> silver stain in over ten years. I want to section the tissue on
> a sliding microtome (freeze the tissue after cryoprotectant) @
> 30um to get nice free-floating sections. The protocol says I
> need to place the sections in formalin (I can use 4% PFA) - store
> the sections in formalin. Can you recommend any other tips to
> this stain -
Follow the instructions to the letter, and if it doesn't work
vary only one thing at a time. Remember that if you have to
move the free-floating sections from one solution to another
there is no intervening water rinse unless it's explicitly
specified in the instructions.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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