Fwd: [Histonet] Problems with immunohistochemistry on formalin-fixed heart sectio ns
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Gillian,
Sounds as if the tissue is inadequately fixed. When you cut them
in half are they rinsed in Krebs, washing off the blood, or just dropped
straight into fix. If into fix do you give several rinses of fix to remove
the blood? Remember the fix will fix the blood before getting to the
cardiac muscle.
Ian.
>From: "Barlow, Gillian"
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>Subject: [Histonet] Problems with immunohistochemistry on formalin-fixed
>heart sectio
> ns
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>Dear Histonetters
>
>This is the latest installment in our ongoing heart saga, I hope someone out
>there can help!!!
>
>Following advice from you all (many thanks!) we now follow the following
>protocol: we harvest our hearts from live but anaesthetized animals so that
>they are still beating, cut them in half to allow the fixative access to the
>tissues, and fix in 10% buffered formalin for usually 12 to 24 hours at room
>temp. After this, we process, embed in paraffin and section at 8 microns.
>The preservation of the tissue looks good when we stain with H&E, but when
>we do the immunohistochemistry, the cells look destroyed. Commercial
>sections purchased from Novagen, who fix their hearts in 4%
>paraformaldehyde, look fine using identical immunohistochemical techniques.
>Are formalin-fixed hearts known to be more sensitive e.g. to pretreatment
>with peroxide to block endogenous peroxidases? Or is there some other step
>likely to be causing trouble? The antibodies are from Santa Cruz, and are
>supposed to work well on formalin- or PFA-fixed tissues.
>
>All suggestions gratefully received!
>
>Many thanks
>Gillian
>
>Gillian Barlow, PhD
>Postdoctoral Fellow
>Laboratory of Julie Korenberg, PhD, MD
>Cedars-Sinai Medical Center
>Davis Bldg, Lab 2007
>110 George Burns Rd
>Los Angeles, CA 90048
>
>Phone: (310) 423 7650
>Fax: (310) 423 0302
Dr. Ian Montgomery,
Histotechnology,
Graham Kerr Building,
Institute of Biomedical & Life Sciences,
University of Glasgow,
Glasgow,
G12 8QQ.
Tel: 0141 339 8855
Office: 4652
Lab: 6644.
Pager: 07625 702883
e-mail: ian.montgomery@bio.gla.ac.uk
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Gillian,
Sounds as
if the tissue is inadequately fixed. When you cut them in half are they
rinsed in Krebs, washing off the blood, or just dropped straight into
fix. If into fix do you give several rinses of fix to remove the blood?
Remember the fix will fix the blood before getting to the cardiac muscle.
Ian.
From: "Barlow, Gillian"
<Gillian.Barlow@cshs.org>
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Subject: [Histonet] Problems with immunohistochemistry on formalin-fixed
heart sectio
ns
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Dear Histonetters
This is the latest installment in our ongoing heart saga, I hope someone
out
there can help!!!
Following advice from you all (many thanks!) we now follow the
following
protocol: we harvest our hearts from live but anaesthetized animals
so that
they are still beating, cut them in half to allow the fixative access to
the
tissues, and fix in 10% buffered formalin for usually 12 to 24 hours at
room
temp. After this, we process, embed in paraffin and section at 8
microns.
The preservation of the tissue looks good when we stain with H&E, but
when
we do the immunohistochemistry, the cells look destroyed.
Commercial
sections purchased from Novagen, who fix their hearts in 4%
paraformaldehyde, look fine using identical immunohistochemical
techniques.
Are formalin-fixed hearts known to be more sensitive e.g. to
pretreatment
with peroxide to block endogenous peroxidases? Or is there some
other step
likely to be causing trouble? The antibodies are from Santa Cruz, and
are
supposed to work well on formalin- or PFA-fixed tissues.
All suggestions gratefully received!
Many thanks
Gillian
Gillian Barlow, PhD
Postdoctoral Fellow
Laboratory of Julie Korenberg, PhD, MD
Cedars-Sinai Medical Center
Davis Bldg, Lab 2007
110 George Burns Rd
Los Angeles, CA 90048
Phone: (310) 423 7650
Fax: (310) 423 0302
Dr. Ian Montgomery,
Histotechnology,
Graham Kerr Building,
Institute of Biomedical & Life Sciences,
University of Glasgow,
Glasgow,
G12 8QQ.
Tel: 0141 339 8855
Office: 4652
Lab: 6644.
Pager: 07625 702883
e-mail: ian.montgomery@bio.gla.ac.uk
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