Re: [Histonet] Antigen retrieval on very old tissue
You realize that an extended fixation times in formalin are damaging
to many antigens, secondary to the induction of molecular cross-links in
Since your (archival) specimen has been in formalin for 20 years your
difficulty has increased dramatically since now you need to do IHC on this
critical specimen. However, I don't think problem is unusual. I'm sure
others doing IHC on archival tissues for various studies.
You didn't mention if this was a whole brain and from what species? Do
if the fixative is really (10%) NBF? Has the fixative ever been changed
period? What type of IHC do you plan to do? Free-floating sections or
sections? Even if you don't know the answers to all these question, I
by removing the brain from the formalin and place in a container with
tap water and
wash the specimen in running water for a long period of time. I would
start with a
3-4 week washing or longer (Puchtler & Meloan 1985 "On the Chemistry of
Formaldehydr Fixative & its Effects on IHC Reactions, Histochemistry 82:
Also Julies Elias, The Journal of Histotechnology Vol.24, No. 3 2001.)
This is the
simpliest non-heating method of AR. Elias 1990 adopted this method
storing de-paraffinized sections in 10% sucrose in PBS at 4C for 16
immunostaining. Subsequently, it's shown that some chemicals, such as
or borohydride in water, may improve the retrieval of masked antigens
Costa et al, 1986, Hausen & Dreyer 1982.) In your case your will have to
"test battery" with different times.
In fact, I would suggest that you do a "test battery" on every critical
level of IHC.
These conditions will give you an optimal protocol for your IHC (and)
even if nothing
works you'll know you tried a whole gamut of things to make it work! I
different AR solutions: citrate buffer, Tris-HCl buffers or even a good
pH 1-4, pH 6-8, pH 10-11. Try different heating methods: microwave (MW)
MW & pressure cooker...maybe low heating or shorter heating times. As
crosslinkages are complicated processes that depend on a variety of
temp, conditions of tissue & fixation that lead to a variety of protein
you need to think about which detection system is best for your needs
and the factors
that deal with this system.
I hope this helps some.
Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
> What is your recommendation for doing AR on very old brain tissue, while
> minimizing damage?
> I have a brain that has been floating in formalin for 20 years,
> and need to do immuno staining on it.
> Bob Nienhuis
> Neurobiology Research
> UCLA / VA Medical Center
> Los Angeles, CA
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