RE: [Histonet] tissue separating from OCT

From:"Monfils, Paul"

I don't know what causes this sporadic problem, but I have experienced the
frustration of it a number of times, primarily with brain specimens that
have been frozen in OCT by someone else (I work in a research service lab
and you never know what someone will bring in the door).  For brains that I
freeze myself, I have generally been able to avoid the problem by soaking
the cryoprotected specimen in liquid OCT for 15 minutes before freezing it
(I use scintillation vials for this purpose).  Once a brain is frozen in OCT
though, I have found no way of getting around the problem when it is
present, other than to thaw it, soak it in OCT, and refreeze it.

> ----------
> From: on behalf of
> Sent: 	Friday, June 23, 2006 4:19 PM
> To:
> Subject: 	[Histonet] tissue separating from OCT
> Hello Histonetters:
> I am having difficulty sectioning this mouse brain.  The tissue is
> separating
> from OCT upon sectioning.  Let me give you a brief background of how the
> brain
> was processed.  Perfused with 4%PFA, fixed overnight at 4C(same fix), next
> day
> rinsed with 1XPBS, then serially cryoproctected overnight with 20%, 30%,
> 40%
> sucrose/1XPBS - tissue sunk every time.  Rinsed tissue from the sucrose
> with
> 1XPBS, blot dry, then flash froze in OCT, immersed in isopentane @ -80C. I
> am
> trying to section at 10um.  I've done this procedure before, many, many
> times,
> never had problem trying to get a nice section.  I am about to pull my
> hair
> out!!!  What's the point in putting a tissue that has wrinkles on a slide.
> I
> don't know what my problem is!    I've adjusted the angle on the cryostat
> &
> adjusted the temperature, changed blades, changed the position of the
> tissue to
> make almost a full circle(cryostat specimen holder).  Can someone in
> histoworld
> help with this frustration?  I thought I was just having a bad cutting
> day, but
> this is now carried on for over a week.  I need to section the entire
> brain -
> ever piece is precious to me.
> Helpless,
> Dina
> Dina D. Gaupp, B.S., M.T.
> Medical Research Specialist
> Center for Gene Therapy
> Tulane University Health Sciences Center
> JBJ Bldg/Rm 658, SL-99
> 1430 Tulane Avenue
> New Orleans, LA 70112
> Lab: 504.988.1194
> Fax: 504.988.7710
> Email:
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