Hey Susan and Confocal Folks --

        The extractive fixations usually used for localizing microtubules
(MTs) are meant to get rid of the soluble form of tubulin, which interferes
with trying to localize the polymerized tubulin (the tubules).  You might
try antibodies against acetylated alpha-tubulin or tyrosinated tubulin to
visualize at least the subclasses of MTs enriched in those modified forms
of tubulin, with little binding to non-polymerized forms.  Sigma sells the
monoclonal, they worked well for me on formaldehyde-fixed tissue.

Greg.

>Does anyone have a protocol for tubulin staining in cells transfected with a
>GFP-fusion protein.  We see a cytoplasmic localization of the GFP fusion
>protein, and it looks filamentous similar to tubulin.  We would like to stain
>for tubulin with a red fluorescent probe so that we could check for
>co-localization.  I'm worried about losing our GFP signal during the fixation
>needed for tubulin staining.  Most of the protocols that I have seen using
>monoclonal anti b-tubulin require fixing in methanol, ethanol or acetone which
>destroys the GFP.  We tried the red-orange fluorescent BODIPY 564/570
>paclitaxel
>from Molecular Probes, but we didn't routinely get a filamentous pattern.  Any
>help and/or suggested protocols would be greatly appreciated.
>
>Susan Garfield
>Laboratory of Experimental Carcinogenesis
>Division of Basic Sciences
>National Cancer Institute, NIH
>Building 37, Room 3C28
>37 Convent Drive MSC4255
>Bethesda, MD  20892-4255
>Phone: 301-496-5688; Fax: 301-496-0734


Greg Martin
Light/Confocal Microscopy
Biological Imaging Service
The Jackson Laboratory
TJL Box 43
600 Main Street
Bar Harbor. ME  04609

207-288-6310 or 6321