[Histonet] Re: Wrinkles on Glutaraldehyde Fixed Tissue

Sat May 10 01:26:50 CDT 2014

Hi Tony,

Your statement makes sense‹glutaraldehyde is quick to fix but slow to
penetrate, and I realized that when comparing the tissue fixation time
frames I used (24h and 48h or more). The tissue that was fixed for 24h is
very hard to spread in the water bath; on the other hand, that which was
in the fixative 48h or longer spread out nicely. Hence, the 24h tissue may
have gotten underfixed, making it difficult to spread and become
wrinkle-free.

Ricardo

On 5/8/14 6:17 PM, "Tony Reilly" <Tony.Reilly <@t> health.qld.gov.au> wrote:

>Hi Bea
>
>The problem with Glutaraldehyde is not its fixation but it's penetration
>rate.  It is very slow to penetrate which is why EM tissue specimens must
>be diced to such small pieces.  If you want to use Glut you will need to
>cut the tissue very thin and use the extended fixation time.
>
>Regards
>Tony
>
>
>Tony Reilly B.App.Sc,  M.Sc
>Chief Scientist
>Anatomical Pathology
>Pathology Queensland PAH
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>Health Services Support Agency| Department of Health
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>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Ricardo
>Leyva
>Sent: Friday, 9 May 2014 4:16 AM
>To: Tony Reilly; Bea DeBrosse-Serra; histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] Re: Wrinkles on Glutaraldehyde Fixed Tissue
>
>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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>>Histonet mailing list
>>Histonet <@t> lists.utsouthwestern.edu
>>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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