[Histonet] Antigen retrieval on very old tissue

[Maria Mejia]

Dear Bob,

You realize that an  extended fixation times in formalin are damaging
to many antigens, secondary to the induction of molecular cross-links in 
proteins.
Since your (archival) specimen has been in formalin for 20 years your 
level of
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 
there are
others doing IHC on archival tissues for various studies.

You didn't mention if this was a whole brain and from what species? Do 
you know
if the fixative is really (10%) NBF? Has the fixative ever been changed 
during this
period? What type of IHC do you plan to do? Free-floating sections or 
paraffin
sections? Even if you don't know the answers to all these question, I 
would start
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: 
201-204.
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 
routinely by
storing de-paraffinized sections in 10% sucrose in PBS at 4C for 16 
hours (before)
immunostaining. Subsequently, it's shown that some chemicals, such as 
urea, acid,
or borohydride in water, may improve the retrieval of masked antigens 
(Larson 1988,
Costa et al, 1986, Hausen & Dreyer 1982.) In your case your will have to 
do a
"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 
would try
different AR solutions: citrate buffer, Tris-HCl buffers or even a good 
commerical at
pH 1-4, pH 6-8, pH 10-11. Try different heating methods: microwave (MW) 
alone,
MW & pressure cooker...maybe low heating or shorter heating times. As 
you know
crosslinkages are complicated processes that depend on a variety of 
conditions: pH,
temp, conditions of tissue & fixation that lead to a variety of protein 
alteration. Then
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.

Yours

Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
Email: maria <@t> ski.org
Phone: (415)-345-2185



nienhuis <@t> ucla.edu wrote:

>
> 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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