Re: Calling all "eye" experts

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From:Barry Rittman <>
To:histology <>
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                   I don't work with eyes very much and am ceratinly not an
expert but have worked extensively  with epithelia.

How much of the retina do you wish to retain?
The attachment of the retina is not always a tenacious one. You may  try cutting
around behind the ora serrata and try pulling the retina from the underlying
connective tissue of the uveal layer.
As the retina has a distinct basement membrane, you can probably completely
separate the retina from the uveal layer by using disodium EDTA to remove the
calcium. This is a standard technique for separating epithelia from the
underlying connective tissue by removing calcium which is critical  for
maintaining adhesion of the basement membrane to the connective tissue.
We have used this technique for several years to separate epidermis from the
dermis in mouse and rat  skin and also for separating  epithelia in mucosae. This
is also a common technique in the steps toward preparing epithelia cel
suspensions as cells remain vital during this separation.
We used  a 3 mM soln. of disodium ethylene diammine tetra acetic acid in 0.1M
phosphate buffered saline pH 7.3 at 37 C for 1-2 hours. For epithelia that resist
separation,  we have used up to 10mM but would recommend using 3mM as a first
attempt. Following this the epithelium can be gently removed by pulling with fine
forceps and then can be fixed , or subjected directly to IHC. No apparent
difference in IHC staining for several of the antibodies that we used.
call me if you need further details

Angela Nista wrote:

> Good Morning!
> I was hoping that someone out there was familiar with doing eye dissections.
> More specifically, I would like to know if anybody has any good reference
> materials on how to separate the retina from the rest of the eye for a flat
> mount, to be analyzed by fluorescence microscopy.  We will be doing this in
> adult rats and mice to be followed by prenatal specimens of the same species.
> Thanks in advance for the info!
> Angela Nista, MS, HT(ASCP)
> Bayer Corporation
> West Haven, CT
> (203) 812-5001

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