[Histonet] Re: Wrinkles on Glutaraldehyde Fixed Tissue

[Ricardo Leyva]

Hi Bea,

Thank you for your response. The reason to 4% Glut use is just a PI
request (really a matter of preference), and it is only on tissue that
will not be immunostained. The ocular tissue I work with is rabbit. One
thing I noticed is that the tissue that is fixed for 24h takes longer to
become wrinkle-free, whereas the 48h (or more) fixed tissue seems to
spread out in the waterbath with better consistency.

I am aware of the use of Davidson's‹in fact you are the one who so
patiently gave me recommendations on it, and for that I am grateful.
Nonetheless, something I'd encounter with Davidson's each time is that
tissue would shrink a bit too much after processing (all of my
reagents‹EtOH, Xylenes, and Wax are set for about 1h20min each); I tried
decreasing the processing time but the issue remains. Perhaps the
processing time needs to be increased on  my end to get better results.


Ricardo


On 5/7/14 7:13 AM, "Bea DeBrosse-Serra" <BDeBrosse-Serra <@t> isisph.com> wrote:

>Why do you use glutaraldehyde for eyes? Davidson's is a much better
>fixative for eyes. With Davidson's you have two fast penetrating
>ingredients and one slow acting fixative. Glut is only a fast fixative.
>Besides, the best institutions who deal with eyes only use Davidson's.
>Just my thoughts. 
>
>Bea
>
>Beatrice DeBrosse-Serra HT(ASCP)QIHC
>Isis Pharmaceuticals
>Antisense Drug Discovery
>2855 Gazelle Ct.
>Carlsbad, CA 92010
>760-603-2371
>
>
>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Ricardo
>Leyva
>Sent: Tuesday, May 06, 2014 11:38 AM
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] Wrinkles on Glutaraldehyde Fixed Tissue
>
>Dear Histonetter,
>
>I am having an issue getting rid of wrinkles on sections from 4%
>glutaraldehyde fixed paraffin embedded ocular tissue. The laboratory's
>tissue of interest is the retina, and I use glut to avoid retinal
>detachment (this is mainly on tissue that won't be immunostained). I
>normally section at 5µm, and fix the tissue 1 or 2 days. I set the water
>bath at a temperature that is 5-10 C below the wax melting point. I have
>tried adding ethyl alcohol to the water and the wrinkles remain-or takes
>very long for most of them to be gone. I have tried placing the sections
>in a 1:15 mix of ethanol and water-and have also tried 30% ethanol in
>water, both at RT, before placing the sections on the water bath; when I
>do this, the sections separate abruptly (once in the water bath) from the
>slide I use for transferring the tissue, causing tissue layer separation.
>When I work with 10% formalin, the section wrinkles disappear nicely in
>the water bath that contains water and a bit ethanol. Nonetheless, when
>it comes to 4% glut it just does not work.
>
>Any input in troubleshooting this is greatly appreciated.
>
>Best,
>
>Ricardo Leyva
>(858) 822-1629
>rleyva <@t> ucsd.edu
>UCSD Shiley Eye Center
>
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