I am performing experiments for cellular binding of specific peptides. These peptides are labelled with FITC. For some peptides, I do not see any fluorescence in the microscope, however and flow cytometry analysis reveals a significant binding as compared to the control cells. I can understand that this is due to the difference in the sensitivity of the two techniques. Please share your experiences on this issue.
Even with the photobleaching issues that FITC has, you should at least see **something** even if only for a short time...if you are truly seeing absolutely nothing, I would check your filter cube set to ensure you have an optimal ex/em pair. First you might want to try another dye like the Alexa or even Cy-x dyes...is there a specific reason you want to stay with a green dye? Are you using digital imaging to capture the microscope image, and if so, are you using an image analysis software to analyze the binding? With a proper technique, the binding should be the same as shown with flow cytometry, so if you're not seeing that, I would first check the things you can before switching dyes, though.
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