Hi,

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.

My questions are...
1. Am I using too much Cy3?  (I use Jackson's SAv-Cy3 at 1;2,000 dilution.
Stained for 30 min at 4C-degree)
2. Someone told, if I remember correctly, Cy3 is a rather big molecule.  If
this is correct, can  intense washing get rid of the big molecules off from
cells and outer fields?
3. Is there any good red-color alternatives that gives less background?

Any suggetion will be appreciated.

Thank you.
Mari