Shinohara, Mari wrote:

> 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.

Have you ever run the secondary antibody through a freeze-thaw cycle?
If so, that may be the problem.  One freeze-thaw cycle, in my
experience, turns most Jackson secondaries from being superb, to being
rather mediocre.

Good luck!

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                        office: (612) 626 0145
Associate Professor                             lab:    (612) 624 2991
Dept. Neuroscience                      Preferred FAX:  (612) 624 8118
University of Minnesota                 Dept FAX:       (612) 626 5009
Minneapolis, MN  55455                e-mail: [log in to unmask]