Re: staining fresh frozen brain sections

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From:"Barry Rittman" <> (by way of Marvin Hanna)
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Hi Lilith,
                I must disagree with you on this one. IHC can be done on fresh
free floating sections but as with fixation it depends on the substances that
you are trying to demonstrate, type of tissue, thickness of sections and so on.
There are of course drawbacks to both. With fresh sections the tissue may swell
and depending on the length of incubation there may be displacement of
With fixed tissues, some substances if present at low concentrations may be
or masked. Components that are reacting may give false positives after

this is a large topic and there are several things to consider, the above are
just a few.
Barry Rittman

"Barry, Lilith" wrote:

> Hi Cynthia,
> I routinely work on free floating sections. This is on fixed sections
> (30-50microns thick). I do not think that it is possible to do
> immunohostochemistry on fresh floating sections. For that I cryosection
> (10-14 microns), mount them in supperfrost + slides and do the immuno on the
> slides. You can cut thick sections, fix them immediately and then proceed
> with free floating procedure. The immuno is done in tissue culture well
> plares by adding and removing solutions.
> As for the processing,  what kind of secondary are you using? If it is
> fluorescent, then I mount with wet mount. If it is permanent, then
> dehydrate and mount with permount.
> Please feel free to ask any question.
> Lilith
> Lilith Ohannessian-Barry
> National Research Council
> Institute of Biological Sciences
> Tel;613-993-6460
> Fax;613-941-4475
> e-mail; <>

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