[Histonet] Mouse eyes

[gayle callis]

Davidsons fixative is popular for eyes.  This has been discussed at great
length on Histonet, so search the Archives.  I believe it works for IHC.
Gluteraldehyde can crosslink antigens too strongly if IHC is needed.  Retina
commonly detaches, at least with larger eyes we have worked with, when the
eye section is flattened on a water bath.  The water bath temperature used
was a few degrees lower than normally used for other tissues. You should
drain the slides well in upright position if using Plus charge.  Lay flat on
slide warmer, to dry at 37C to 40C overnight or longer as we do for
decalcified bone sections.

There are other ways to flatten eyes by floating an eye section on 5%
alcohol water bath, then picking up on a slide, and slowly lowering the
section (keep top part of paraffin of section attached to slide, and
lowering this into a warm waterbath.  The surface tension is reduced by the
alcohol, allowing the eye to very slowly flatten, then pick up, drain well
then dry flat. This takes a bit of practice but also works for decalcified
bone when the softer cartilage likes to wrinkle versus the harder bone.
Always pays to slow down speed of water rinsing, if the water flows too
hard, during staining.  

Gayle Callis
HTL/HT/MT(ASCP) 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Roger Moretz
Sent: Monday, November 23, 2009 2:15 PM
To: Durden, Kelley; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Mouse eyes

Altho' retired for 2 years, we had a protocol that retained all parts of the
eye quite well.  Fixation was in 3% glutaraldehyde (diluted in H2O), 4 deg.C
for overnight.  Don't extend the fixation as this will cause the tissue to
be too hard.  Wash for about 1hr in running tap water and process as usual
for mouse tissue (if it works for mouse liver, it will work for the eyes).  

Roger Moretz, Ph.D. (ret.)



----- Original Message ----
From: "Durden, Kelley" <kelleydurden <@t> pathology.ufl.edu>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Sent: Mon, November 23, 2009 10:44:21 AM
Subject: [Histonet] Mouse eyes

My question concerns mouse eyes.

Can anyone send me their fixation suggestions?  We are receiving mouse eyes
that end up with a "wavy" look instead of a nicely defined "C" and have no
explanation for this occurrence.

Can anyone send me their mouse eye processing schedule?  We have a protocol
that has proved fast and true but would welcome other suggestions.

Can you give me a good idea for making sure sections stick to slides well? 
Gold Plus?  What else?

Has anyone experienced a retinal detachment after or upon staining?  Retina
attached after sectioning - detached after staining - just routine H&E. 
Should I heat for an extended period of time?

Kelley Durden, HT ASCP
University of Florida
Molecular Pathology Core
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