>Paul,
>We are trying to establish IHC/ISH procedure in our lab, and it looks like
>when I do antigen retrieval, the ISH probes tend to bind non-specifically.
>What is the sequence of your work? when do you perform the retrieval step?
>It would be great if you could e-mail your protocol for the pituitary
>hormones.
>Thanks a lot in advance,
>Anna

Hi Anna,

We perform our double IHC/ISH in the following sequence.

1. Non-radioactive ISH with riboprobes, detected with alcaline phosphatase
labelled antibodies using  NBT/BCIP as chromogens. Chromogen development is
checked under the dissecting scope. The slides are then selected for
further IHC processing. We sometimes store the slides for up to several
months in buffer + preservative before doing the IHC. Slides to be
double-labelled are treated for 5 min with 95% ethanol to shift the violet
NBT/BCIP label to blue. Some signal (and a lot of background) is lost
through this, but usually we get along.

2. IHC. It's at this stage that we tried the antigen retrieval, which
unfortunately never improved things. We do our IHC using standard ABC
techniques with peroxidase or alcaline phosphatase. The alcaline
phosphatase remaining from the ISH step is much lower than that from the
IHC step and never interfered with our experiments. If we use peroxidase,
we usually detect with amino-ethylcarbazole as chromogen. This red
chromogen contrasts very nicely with the violet-blue ISH label, and
double-labelled cells are easily distinguished. We coverslip the slides
after applying a Crystalmount film, which allows alcohol-soluble chromogens
to be mounted with permanent toluene-based mountants like Eukit or DePeX.

We perform most of our work on polyethylene-glycol (aka as PEG or carbowax)
embedded tissue sections. The only thing that improved immunolabel after
the ISH was the use of free floating sections. Some antigens (intermediate
filaments, Pit-1) seem to resist the ISH procedure on free floating
sections and not when slide-mounted sections are used. We never perform the
ISH after IHC, because IHC exposes the sections to RNAses which reduce and
even abolish our ISH signals.

As to our protocol for pituitary hormones, its a straightforward standard
ABC technique with primary antibodies we obtained from Dr. Parlow at the
NIDDK. Many people use those and they do not seem to require any antigen
retrieval whether used on paraffin, PEG or cryosections.

Paul

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Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur
12 rue de l'Universite
F-67000 Strasbourg, FRANCE
tel: 03.88.35.85.04    fax: 03.88.24.04.61
===============klosen@neurochem.u-strasbg.fr==============================