You could try applying Protein-G labelled with a fluorophore to those
sections.  That would identify the antibody even if the FITC had been lost
from the antibody.  If you do not find the antibody this way then you could
permeabilize the section and look for intracellular antibody with the same
technique- just in case the antibody was internalized.
You could also consider using a different fluorophore such as Texas Red
which may be less reactive than FITC- at least it bleaches about ten times
slower than FITC for whatever that is worth.

Milton Charlton
University of Toronto

----- Original Message -----
From: "Christian Broesamle" <[log in to unmask]>
Newsgroups: bit.listserv.confocal
To: <[log in to unmask]>
Sent: Wednesday, October 04, 2000 8:03 AM
Subject: In vivo FITC-Ab halflife


> Not strictly confocal:
> We have tried to track the distribution of a FITC-labelled Ab after
> injection into the cerebellum of P7 rats. We were very dissapointed to
find
> virtually NO fluorescence at 2 days after injection of 1 ul of a standard
> secondary Ab stock solution. The injection was under visual control and we
> were able to identify the needle track on the sections. The animals were
> perfused by ringer followed by formaldehyde (all buffered at pH7.2) and
the
> brains cryosectioned. Could it be that the FITC fluorochrome has been
> degraded so quickly in the animal? Next we will look 2 hrs. after
injection
> but we would also like to know about 2 days. Any comments, suggestions
welcome.
>
> Christian
> *********************************
> Christian Broesamle, PhD
> Brain Research Institute
> Department of Neuromorphology (Gruppe Schwab)
> Federal Institute of Technology and
> University of Zurich
> Switzerland
> [log in to unmask]
>
> Tel: +41 1 635 3216
> Fax: +41 1 635 3303
> Street address:
> Winterthurer Strasse 190
> CH-8057 Zurich
> Switzerland
> *********************************
>