Re: IHC to see degenerative axon fibers

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From:"J. A. Kiernan" <>
To:Maria Mejia <>
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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