Re: Fixation and embedding of adult murine eyes

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From:Carolyn Pressman <> (by way of histonet)
To:histonet <>
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Dear Lynn,
Thank you so much for your prompt reply. I would definitely love to have
the two protocals that you mentioned.  I have another question for you.
What is the advantage of using pen-fix versus neutral buffered formalin?
A post-doc in our lab said that this sort of fixative is better for
preserving extracellular matrix epitopes for immunohistochemistry.  In
addition to doing routine histology (H + E and mallory's trichrome), I
may need to perform section in situ hybridization using RNA probes. This
technique works fine with normal formalin, but do you think it will also
work with the pen-fix?
Please email the protocals, or if you prefer, our fax # is 713 791 9478
Again, thank you so much!
A struggling graduate student,
Carolyn Pressman
MD Anderson Cancer Center
University of Texas Houston
Phone: 713 794-5811

On Tue, 26 Jan 1999, Lynn Gardner wrote:

> Dear Carolyn,
> You came to the right person, mostly what we process is eye tissue from
> human to murine. We find that Pen-fix which is an alcoholic formalin from
> Richard Alan (1-800-781-2307) works very well. I would be happy to send you
> two protocols we use for processing these eyes that work very nicely. One
> is an overnight process and as long as the tissue has fixed for 4 hours
> prior the tissue will do fine. We also have a very quick process which we
> can do. The tissue needs to fix for 4-6 hours and then we run a short cycle
> on the processor the difference is how fast the researcher needs their
> materials. We perform routine, special and immunohistochemistry on our
> tissues and this fixative seems to enhance the staining qualities of the
> tissues and is very versitile.
> As for the lens problem, we have found that if you take a 0.25-0.5%
> ammonium hydroxide (in water) and pour it into a dish containing ice
> carefully trim you block so that the lens is exposed. Place the block into
> the ice/Ammonium hydroxide solution and leave it sit for 15 minutes. Then
> try cutting the tissue this solution will usually hydrate the tissue enough
> to allow the lens to cut beautifully, if not soak the block a little
> longer. If this does not resolve the problem try backing down the times in
> your paraffins, xylenes and 100% alcohols during processing decrease them
> each by 10 minutes to start and see if this helps the situation.
> If you have any futher need of eye or other information please do not
> hessitate to let me know. Good Luck!
> Sincerely,
> Lynn Gardner, HT(ASCP)
> Supervisory Research Assistant III
> FC Blodi Eye Pathology Laboratory
> The University of Iowa
> 233 MRC
> Iowa City, IA 52242-1182
> Phone: 319-335-7095
> Fax: 319-335-7193
> At 11:41 AM 1/26/99 -0600, you wrote:
> >What is the best fixative to use for adult murine eyes?  Does anyone have
> >any good protocals?  I have found through trial and error that fixing in
> >neutral buffered formalin only works ok if I fix for 2 days.  I embed in
> >parafin and then section at 7 um.  Most of the architecture of the eye is
> >intact, but the lens always tears or has holes in it.  Is there a way to
> >get around this problem?  Is embedding in plastic a better way to do this?
> >Carolyn Pressman
> >MD Anderson Cancer Center
> >University of Texas--Houston
> >

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