Just a point on something in Jacques' message about Triton - wehave neve
used Triton or any other detergent in any of our immunolabelling -
wholemounts, cryo sections, PEG sections or Vibratome sections or
preparation for TEM immuno. In our hands, detergents lead to
siginificant degradation of tissue morphology (you should see it at TEM
!!) and lead to a lot of weird "fluorojunk". We usually permeabilise
/clear our tissue prior to sectioning with DMSO and ethanol, which works
a treat with very little generation of artefacts. The absence of
detergents in the antibody mixes allows for really clean labelling with
very small amounts of antibody solution...

Hope that helps

IAN


Jacques Paysan wrote:
> In our
> experience, best results were obtained by incubating the antibody for 30' at
> 15°C in PBS containing 2 - 10% of normal serum from the same species in
> which the secondary antibody was made (plus optional 0.2% Triton).  4.)
> Check whether you are using the correct filter sets.  You should also keep
> in mind that what counts is the signal to background ratio, rather than the
> absolute background.
>
> Particularly the donkey Cy3-conjugated secondaries from Jackson are
> excellent.  You shouldn't have any persisting problems (unless something is
> wrong with your lot).  If problems persist feel free to send your detailed
> protocol and I will check for other possible problems.
>
> Good luck,
> Jacques
>
> ---------------------------------------------------------------
> Jacques Paysan, PhD
> University of Hohenheim
> Institute of Physiology 230
> Garbenstrasse 30
> D-70593 Stuttgart
> Germany
>
> Phone: ++49-711-459.22.67
> Fax:  ++49-711-459.37.26
> email:  [log in to unmask]
> Web:  http://www.paysan.de/lynx.htm
>
> ----- Original Message -----
> From: "Shinohara, Mari" <[log in to unmask]>
> Newsgroups: bit.listserv.confocal
> To: <[log in to unmask]>
> Sent: Friday, August 11, 2000 4:57 PM
> Subject: questions on Cy3
>
> > Hi,
> >
> > I am now trying to see subcellular localization of molecules.  When I use
> > FITC as secondary staining, it looks nice.  However, if I use Cy3, there
> > appears pretty high background (all over, not only in cells) and the whole
> > field looks orangish as well.
> >
> > My questions are...
> > 1. Am I using too much Cy3?  (I use Jackson's SAv-Cy3 at 1;2,000 dilution.
> > Stained for 30 min at 4C-degree)
> > 2. Someone told, if I remember correctly, Cy3 is a rather big molecule.
> If
> > this is correct, can  intense washing get rid of the big molecules off
> from
> > cells and outer fields?
> > 3. Is there any good red-color alternatives that gives less background?
> >
> > Any suggetion will be appreciated.
> >
> > Thank you.
> > Mari

--
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]