Fixation of frozen sections-reply to R.Geske

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From:"Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL>
To:Histonet <>
Date:Fri, 28 May 1999 16:56:56 -0400
Content-Type:text/plain; charset="iso-8859-1"

For all you histonetters, I forgot to hit the "reply to all" button the
first time.

Noelle Patterson
Naval Medical Research Center
Bethesda, Md

From:  Patterson, Noelle
Sent:  Friday, May 28, 1999 4:53 PM
To:  'Rob Geske'
Subject:  RE: Fixation of frozen sections

Remember, the slides are fixed in acetone completely first.  I do quick dips
in water only to rinse off OCT that has a tendancy to fold onto the tissue
if left there.  No air drying between has been necessary since I then go
back into the same acetone as the fixation, 10-20 quick dips, as a means to
"quick dry" the slides after water.  So far, much cleaner slides, great IHC,
and faster "air dry" times.

So, yes I do dips, but not dips into an acetone/water mixture.  The acetone
is tossed/not reused after each batch of slides runs through it.  In other
words, I never use the acetone to "fix" once the water-dipped slides have
been run through it.

I use this for a number of lymphocyte CD and activation antigens/markers.  I
haven't needed to do any other type of IHC.  The water steps are not long
enough to cause diffusion.

Noelle Patterson
Naval Medical Research Center
Bethesda, Md

	From:  Rob Geske []
	Sent:  Friday, May 28, 1999 4:10 PM
	To:  Patterson, Noelle
	Subject:  RE: Fixation of frozen sections


	did you really mean "........(10-20 dips in water then
acetone)....."?  do you thoroughly dry your slides after the water and
before the acetone? i have always understood that acetone contaminated with
water could have an ill effect on immunoreactivity of "some"
antigens...........maybe not yours eh?


	At 12:40 PM 5/28/99 -0400, you wrote:
	>I asked a similar question to histonet not too long ago.  I got
enough ideas
	>to run my own study.  What I can tell you is that monkey and human
	>tissue are best when sectionned, fixed (I use Acetone at -20C), air
	>(about 15-30 min., the minimum to get dry slides), and stored at
-70C until
	>use.  When I remove the slides for IHC, I do a brief fix (10-20
dips in
	>water then acetone) for reasons based on what our staining system
	>However, murine renal, pancreas, spleen, and lymph node tissue do
just fine
	>if you fix in acetone, air dry, and store (dust-free) at room
	>There are quite varied protocols to address your question.  Good
luck.  Feel
	>free to ask ant nagging questions you may have about what I've
	>Noelle Patterson
	>Naval Medical Research Center
	>Bethesda, Md
	>   ----------
	>   From:  Coskran, Timothy M
	>   Sent:  Thursday, May 27, 1999 3:34 PM
	>   To:  ''
	>   Subject:  Fixation of frozen sections
	>   Does anyone have any ideas on whether it is better to cut
	>fix and
	>   store the sections or to cut frozens, store, and then fix when
	>ready to
	>   carry out a stain?  We currently cut frozen sections, dry
	>and then
	>   store the slides at -80C until we are ready to do an immuno
	>Then we
	>   bring the slides to room temp, fix and proceed with the stain.
	>   some literature that suggests fixing the slides immediately
	>they have
	>   been sectioned and then storing in PBS until ready to use.
	>   If anyone has a basic protocol for frozen sections, would they
	>willing to
	>   share?
	>   Thanks
	>   Tim Coskran
	>   Pfizer
	Robert S. Geske
	Research Associate
	Center for Comparative Medicine and Department of Pediatrics
	Baylor College of Medicine

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