[Histonet] immunohistochemistry and frozen sections

[Phillip Huff]

First, I would recommend that you run some Western Blots to ensure that
your Antigen of interest is being expressed within your tissues. Once your
Western blots are positive, you can definetly say that your immunoreaction
is what needs to be optimized.

Second, have you applied your immunoreaction technique to a known positive
tissue source, that has also been sectioned at 5 um? It may be a good
confirmation that your tissue may need to be processed differently or cut
thicker.

Finally, if your end goal is immunolocalization and the immunohistochem is
not working, you may want to look into in situ hybridization. In your
tissue samples, the receptor may be quite sensitive to processing and you
may be losing a great deal of your antigen. I know very little about your
receptor, but does it have a high turnover rate? Perhaps it has been
degraded over time when frozen. Has the reaction been done in freshly
harvested tissues? Perhaps localizing the mRNA in the tissues may provide
you with the results you are looking for. Before doing this though, I
would confirm that your tissues express the mRNA of interest by doing some
RT-PCR. This will both confirm that your tissues have your target mRNA,
and if you use an internal housekeeping primer in the PCR, you can
determine the approximate copy number of your mRNA to help you in
determining how sensitive your in situ reaction must be.

Hope this helps you out,

Phil



> Hi Histonetters,
>
> I've been struggling for 4 months on my 5 microns
> frozen sections trying to find estrogen receptors.
> I've tried both immunoperoxidase kit from Vector
> laboratories either indirect immunofluorecsence (FITC
> Goat anti mouse Secondary Ab from Sigma) but I've not
> yet obtained good results.
> My primary Ab is a mouse monoclonal to bovine
> Estrogen receptor from Cymbus biotechnology.
> Sections come from tissue that is supposed to be a
> non-target tissue (mid laminar region from cow's
> hooves)blocking with 2% goat normal serum (sigma)
> whash in tbs buffer
> All different methods for antigen retrieval have been
> tested but sections look poor regarding histologic
> quality and I still have very big problems with non
> specific binding an staining (indirect
> immunofluorescence).
> It looks like frozen sections are too tender to
> efford the whole processing.
> Every suggestion or reference is welcome,
> thank U
>
> La Manna Vincenzo
> Department of Agriculture and Forestry, University of
> Aberdeen, 581 King Street, AB24 5UA, Aberdeen , UK.
> Telephone; 01224 274259
> Fax; 01224 273731
> e-mail v.lamanna <@t> abdn.ac.uk
>
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>