Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

We've sometimes used a procedure similar to Mike's below to label
receptor proteins. For this procedure we use cryostat sections rather
than our usual PEG sections. When it works it is great, but we find a
lot of variability even across different regions of the same section,
which can make life pretty difficult at times!

Good luck is definitely required!

IAN


"Michael C. Adams" wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I used to rotinely perform antigen retrieval on frozen tissue sections that
> were fixed with paraformaldehyde using 10mM NaCitrate for two short rounds in
> the microwave (enough to heat the solution without letting it boil) at the
> beginning of the IF procedure.  This seemed to work quite well for IF of
> certain molecules.  I'm not sure about the exact mechanism of the reaction but
> I think the hot NaCitrate breaks some of the cross-bridges formed during
> formaldehyde fixation.
>
> Best of luck!
>
> Mike
>
> >Milton Charlton wrote:
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> Dear confocal list members:
> >>
> >>         In an immunocytochemistry experiment I am having trouble with
> >> antibodies for a particular protein that we expect to be present.  We have
> >> tried several antibodies for this particular protein using paraformaldehyde
> >> or Histochoice fixation with minimal concentration of Triton X or even no
> >> detergent.  We also tried incubating the tissue with the primary antibody
> at
> >> 40C.  Nothing works.    We get good immunoreactivity for other proteins
> >> using other antibodies.
> >>         There are many references to Antigen retrieval using non
> >> physiological pH, chemical treatment or high temperature before application
> >> of primary antibody.  Has anyone found these tricks beneficial when applied
> >> to non-embedded tissue or cells (no wax)?
> >> Thanks for your advice.
> >>
> >> Milton P. Charlton, Professor
> >> Physiology Department
> >> Medical Science Building RM.# 3308
> >> University of Toronto
> >> 1 King's College Circle
> >> Toronto, ON, M5S1A8
> >> Canada
> >> tel:  416-978-6355
> >> fax:  416-978-4940
> >> [log in to unmask]
> >>
> >
> >
> >--
> >Edwin D. Moore, Ph.D.
> >Department of Physiology
> >University of British Columbia
> >2146 Health Sciences Mall
> >Vancouver, V6T 1Z3, B.C.
> >
> >phone:  604-822-3423 or 7719
> >FAX:  604-822-6048
> >email:  [log in to unmask]
>
> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
> Michael C. Adams
> Research Technician for Dr. Clare Waterman-Storer
> Laboratory of Cell Motility Studies
> Department of Cell Biology
> The Scripps Research Institute
> 10550 North Torrey Pines Road, CB 163
> La Jolla, CA 92037
>
> TEL 858.784.9244
> FAX 858.784.7521
> EMAIL [log in to unmask]
> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

--
Professor Ian Gibbins
Anatomy & Histology
Flinders University of South Australia
GPO Box 2100, Adelaide, SA 5001
Australia

Phone:  +61-8-8204 5271
FAX:    +61-8-8277 0085
Email:  [log in to unmask]