Guofeng,

For double immunofluorescence (IF) I first troubleshoot each antibody
with peroxidase, and then use the same conditions (pretreatment,
dilution) for IF. The trick is that both antibodies have to work well
with the same pretreatment. This is not always possible. I would
strongly recommend Alexa fluorochromes from Molecular probes instead of
Texas Red, FITC etc. They are very stable and strong. Molecular probes
carries all kind of colors so that you can match them to what filters
you have available on your microscope. Also, they have them conjugated
to immunoglobulins from various species. You can get donkey
anti-mouse,-rabbit,-goat, etc. Then you can use normal donkey serum to
block.  
Good luck,

Sarka Lhotak
McMaster University
Hamilton, Ontario, Canada

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