Re: double IHC/in situ

From:Marek Malecki <>

Dr. Gwen Childs wrote the whole chapter on the subject in 1995: Specimen 
Preparation for Microscopy and Microanalysis.  SM International Press, 
Chicago, IL.
In addition, her www site is loaded with protocols and images.

> >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:

Marek Malecki, M.D., Ph.D.
Director and Principal Investigator

Molecular Imaging Laboratories, University of California, San Diego
address: 1500 Suite Bonner Hall, 9500 Gilman Drive, La Jolla, CA92093-0368
phone: 8588223373
fax: 8584573355
pager: 8586161420
www site:
ftp site:

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