RE: staining fresh frozen brain sections

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From:"Barry, Lilith" <Lilith.Barry@nrc.ca> (by way of Marvin Hanna)
To:histonet@histosearch.com
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Dear Dr. Rittman,

Thank you for the comment on fresh - floating tissue processing.  Could you
please give some references on this topic. Is it applicable for neuronal
tissue ? What about the  morphology?  I have mainly worked with brain
tissue, labeling neuropeptides  for light and electron microscope and
haven't  come across a paper that deals wit it.

Waiting for your usual informative response,


Lilith
- - - - - - - - - - - - - - - - - - - -

Lilith Ohannessian-Barry
National Research Council
Institute of Biological Sciences
CANADA
Tel;613-993-6460
Fax;613-941-4475
e-mail; lilith.barry@nrc.ca <mailto:lilith.barry@nrc.ca>


		-----Original Message-----
		From:	Barry Rittman [mailto:brittman@mail.db.uth.tmc.edu]
		Sent:	Thursday, January 27, 2000 9:46 AM
		To:	histology
		Subject:	Re: staining fresh frozen brain sections

		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
		components.
		With fixed tissues, some substances if present at low
concentrations may be lost
		or masked. Components that are reacting may give false
positives after fixation.

		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
		> CANADA
		> Tel;613-993-6460
		> Fax;613-941-4475
		> e-mail; lilith.barry@nrc.ca <mailto:lilith.barry@nrc.ca>





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