[Histonet] detached retinas

[Elizabeth Chlipala]

Tyrone

I'm not sure if this will be helpful or not but we do a lot of work with mouse and rat eyes.  We have seen that with both 10% NBF and 4% PFA there will be retinal detachment regardless of how you process the samples.  Fixation in modified Davidsons or Davidsons in our experience is the only way that we are able to maintain good morphology of the retina without it detaching.  I'm not sure how that will affect the fixation of the entire fish, since I do not have experience in processing fish samples.  We have also found that you cannot process the eyes whole if you want any decent retina morphology, for us since we are just dealing with eye samples and not an entire fish, we will trim the edge off of both the rat or mouse eyes that we are running retinal studies on.

I hope this helps.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz <@t> premierlab.com
www.premierlab.com

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tyrone Genade
Sent: Friday, August 29, 2014 11:12 AM
To: histonet
Subject: [Histonet] detached retinas

Hello,

I'm having a problem with detached retinas.

I fix my fish, whole, in 4% PFA for 24 hours. The belly is slit to allow better penetration of the PFA. The entire fish is about 4 cm long and about
6 mm wide. From the 4% PFA I proceed to decalsify the fish in 14% EDTA (pH 7.6). This goes on for 3 days. From there the tissues is passaged through 70% to 95% to 100% ethanol and then cleared in methylbenzoate before being wax impregnated.

My current hypothesis is that the thick eye membrane is impeding the penetration of the PFA. As consequence, when I passage into the EDTA and alcohol I get rapid dehydration of the tissue and the change in pressure in the eye causes the detached retinas. How feasible is this hypothesis and what others can you come up with?

I am currently testing this hypothesis. I have fixed 2 fish in PFA but I have punctured the eyes of one of the fish to improve PFA penetration.

The sections I have cut of the first specimen show no issues with soft tissue fixation. My only problem is that the retina has detached formed several waves of tissue in the eye. The sections look good except for the eye issues. I never had this issue with Bouin's.

Thanks

--
Tyrone Genade

P.S. Incidentally, I am now using Paraplast Plus cat number 19216 ( http://www.emsdiasum.com/microscopy/products/histology/embedding.aspx ) and it is a dream to work with compared to what I was using. It is soft and cuts beautifully, the ribbons look good and impregnation is great (even by manual processing).

Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org
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