Fixation of frozen sections-reply to R.Geske
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From: | "Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL> |
To: | Histonet <HistoNet@Pathology.swmed.edu> |
Reply-To: | |
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
pattersonn@NMRIPO.NMRI.NNMC.NAVY.MIL
----------
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
pattersonn@NMRIPO.NMRI.NNMC.NAVY.MIL
<mailto:pattersonn@NMRIPO.NMRI.NNMC.NAVY.MIL>
----------
From: Rob Geske [SMTP:rgeske@bcm.tmc.edu]
<mailto:[SMTP:rgeske@bcm.tmc.edu]>
Sent: Friday, May 28, 1999 4:10 PM
To: Patterson, Noelle
Cc: Histonet@pathology.swmed.edu
<mailto:Histonet@pathology.swmed.edu>
Subject: RE: Fixation of frozen sections
Noelle,
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?
Rob
At 12:40 PM 5/28/99 -0400, you wrote:
>Tim,
>
>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
renal
>tissue are best when sectionned, fixed (I use Acetone at -20C), air
dried
>(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
needs.
>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
temperature.
>
>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
written.
>
>Noelle Patterson
>Naval Medical Research Center
>Bethesda, Md
>pattersonn@NMRIPO.NMRI.NNMC.NAVY.MIL
>
>
> ----------
> From: Coskran, Timothy M
[SMTP:timothy_m_coskran@groton.pfizer.com]
<mailto:[SMTP:timothy_m_coskran@groton.pfizer.com]>
> Sent: Thursday, May 27, 1999 3:34 PM
> To: 'Histonet@pathology.swmed.edu'
> Subject: Fixation of frozen sections
>
>
> Does anyone have any ideas on whether it is better to cut
frozens,
>fix and
> store the sections or to cut frozens, store, and then fix when
your
>ready to
> carry out a stain? We currently cut frozen sections, dry
briefly
>and then
> store the slides at -80C until we are ready to do an immuno
stain.
>Then we
> bring the slides to room temp, fix and proceed with the stain.
I've
>seem
> some literature that suggests fixing the slides immediately
after
>they have
> been sectioned and then storing in PBS until ready to use.
>
> If anyone has a basic protocol for frozen sections, would they
be
>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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