Re: floating sections

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From:"R.Wadley" <>
Date:Wed, 14 Jul 1999 10:37:37 +1000
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	Dear Sharon,
	Some thoughts for your colleague.

	Formaldehyde is a fixative that works by crosslinking proteins.  Therefore
if 2 sections overlap I am not surprised that they might stick together.
Gentle aggitation may be an answer here, but I suspect that works better on
bulkier specimens.  Fixing individually is the ideal solution, but may be a
pain if you've got dozens of the things.  After fixing & then washing I
suspect the sticking problem may be solved.

	One thing I tried in a research lab I worked in was to interleave thin
specimens with cigarette or slide papers.  This keeps everything separated
until your past the fixation stage, & allows you to process many more
samples at the one time.  Its fiddly making the little parcels but does
allow you to ID each section if you wish.  Once you have the knack its all
relatively quick.

	I suspect that a section fixes much more rapidly than a normal biopsy &
wonder if the fixation time could be reduced to about 1 hr?



At 12:19 PM 7/13/99 -0700, you wrote:
>Dear Histonetters,
>I recently had a colleague ask a question I could not regarding
>fresh frozen floating sections.  I'm passing it on to you in hopes that
>you have more experience with floating sections than I do.  Any
>suggestions or insight would be greatly appreciated.
>I would appreciate if you could give me some advice (since I have tried
>it once but... )
>My problem is that I was not able to perfuse the experimental animals. I
>therefore have to do it on fresh frozen tissue. For what I read, people
>usually collect the sections on slides and process to the in situ. I am
>currently working on finding a way of fixing the tissue to be able to
>on free-floating sections. I believe the sensitivity would be greater
>and it would be easier!
>I have tried different things. I (and a technician in my lab)
>collected sections in :
>- 0.5% paraformaldehyde or,
>- PBS 1M or,
>- CBS (glycerol-etc...)
>The sections were rinced once (and still looked fine).
>Then, we fixed the sections for 2hrs in 4% Paraformaldehyde.
>That's when it went wrong!!!! The sections sticked to each other and it
>was impossible to work with them.
>Do you have any idea of what went wrong? of what I should do to make it
>Have you tried to work on free-floating sections from fresh frozen
>Do you know if people have tried before? Do you think that it's even
>possible or am I wasting my time?
> I would really appreciate your help.

R. Wadley, B.App.Sc, M.L.S
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) 	+61 (2) 9385 3517
Ph (AH)	+61 (2) 9555 1239
Fax 	+61 (2) 9385 1591

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