Susana,

GFP fluorescence is sensitive to organic solvents. Try to use a better
paraformaldehyde protocol for fixing the microtubules, perhaps as described
below. Some new fluorescent proteins are supposed to be more resistant to
solvent effects but I have not tried yet.


Regards, Kirk


-----Original Message-----
From: Confocal Microscopy List
[mailto:[log in to unmask]]On Behalf Of Garfield, Susan
(NCI)
Sent: Wednesday, March 07, 2001 9:56 AM
To: [log in to unmask]
Subject: Re: immunofluorescence MetOH GFP


Susana,

We used both paclitaxel, BODIPY 564/570 (Molecular Probes, P-7501) and
tubulin antibody(Boehringer Mannheim) to label tubulin in GFP transfected
cells.  Cells were fixed in 4% paraformaldehyde prepared in microtubule
stabilizing buffer (80 mM PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl) for 20 min at
room temp.  They were permeabilized in 0.05% Triton X-100 for 5 min at room
temp.  Please refer to Mertts et al., Identification of the Region in the
N-terminal Domain Responsible for the Cytoplasmic Localization of Myoc/Tigr
and Its Association with Microtubules, Laboratory Investigation, Vol. 79,
No. 10, p. 1237, 1999.

Susan Garfield
Confocal Facility Manager
Laboratory of Experimental Carcinogenesis
Division of Basic Sciences
National Cancer Institute, NIH
Building 37, Room 3C28
37 Convent Drive MSC4255
Bethesda, MD  20892-4255
+1 (301)496-5688 x227 (voice); +1 (301)496-0734 (fax)


-----Original Message-----
From: Susana Castel [mailto:[log in to unmask]]
Sent: Wednesday, March 07, 2001 8:42 AM
To: [log in to unmask]
Subject: immunofluorescence MetOH GFP


Dear confocalist,

I'm trying to make immunofluorescence of Tubulin in GFP transfected
cells. I have a good staining fixing with cold methanol but I loose the
GFP signal. With PF the tubulin staining is not so good. Any suggestion?



Susanna