Re: double IHC/in situ (Paul Klosen)

>Hello Histonetters,
>Some of the researchers around here do in situ hybridization followed
>by IHC.  However, the harsh conditions of the in situ hybridization
>techniques destroys many of the antigenic sites of interest.  Does
>anyone out there use antigen retrieval to recover antigenicity
>following in situ techniques, or does anyone know of any references?
>Karen Larison
>University of Oregon

Hi Karen,

ISH techniques sometimes work like "antigen retrieval techniques" for some
antigens. I have used ISH in combination with ICC for pituitary hormones,
and the ICC combined to the ISH was often stronger and with less background
than alone. However, for other antigens, the ISH procedures seem a bit too
harsh, and no antigen retrieval will recover this.
We've tried to combine ISH with ICC for vasopressin, and the immunolabel
was strongly reduced. I suspect that for small peptides like vasopressin,
the proteinase K step combined with the high temperatures of the ISH result
in elutin of some of the peptide.
We have tried microwave antigen retrieval for vasopressin and other
antigens like intermediate filaments, Pit-1, thyroid hormone receptors and
several other antigens, but nothing would do for any of those. I think you
will have to try, but I suspect that antigen retrieval will be of little
help after in situ.


                         (o -) O
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:    fax:

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