On Thu, 23 Feb 1995 11:44:23 -0800, Craig Daly wrote:
 
>Hi Folks,
>
>I have a couple of results which I am having trouble explaining and I hope
>someone can put me right.  I use live tissue (blood vessels and smooth
>muscle cells) on our confocal and recently have been using the probes
>Hoechst 33342 (UV,nuclei), FITC-BSA (extracellular) and BODIPY FL-prazosin
>(an alpha1-adrenoceptor ligand).  When I use Hoechst with either FITC or
>BODIPY I often observe nuclear staining in the 488/515 range.  From the
>mole probes handbook it would appear that the FITC and BODIPY may be
>binding to thiolated nucleic acid.  My questions are;
>
>i.      Why do I only see this when I have double labelled with Hoechst and
>a                   Fluorescein.
>ii.     How does nucleic acid become thiolated.
>iii.    How does BODIPY and FITC get into live cells (receptor
>internalisation or membrane transport?) both probes are supposed to be
>impermeant.
>
>If you are still reading this, do you use Fluorescent ligands for
>receptors?  If so are you interested in receptor localisation and
>pharmacology?  We are trying to develop a method of real time ligand
>binding using fluorescent probes for receptors and would like to make
>contact with anyone who has a similar interest.
>
>Thanks for taking the time to read this.
>
>Craig.
>
>
>
>------------------------------------------------------------
>Craig J. Daly
>Research Associate,
>CRI Heart Failure,
>Institute of Physiology,
>University of Glasgow,
>Glasgow G12 8QQ                 Tel. 44 41 339 8855 ext 6606
>                                Fax. 44 41 330 4100
>                                [log in to unmask]
>------------------------------------------------------------
 
I can't help you with the problems you mentioned (but FITC and BODIPY
shouldn't get into cells unaided)... We also are interested in trying to
localise receptors directly using labelled liagands - so far we've had
little success, manily due to all sorts of background and non-specpfic
binding problems...
Ian Gibbins
Department of Anatomy and Histology
Flinders University of South Australia
Phone:  +61-8-2045271
FAX:    +61-8-2770085