I am currently having issues with my PeCy7 bleeding into other channels on the imagestream. I’m using it to stain for the surface marker CD11b. I have attempted multiple dilutions as well as turning down the voltage on both the 488 and 561 lasers but I still get bleeding into the PE channel as well as the AF647 channel. I even bought new antibodies! All suggestions are welcome!
I personally think it is the reason for the controls. You need to check if your negative control, single-positive control, and double-positive control are correct before you calibrate the compensation. In addition, when selecting fluorescent dyes, try to select dyes with less overlapping emission spectra to avoid spillover.
I think if you compensate well this should not be an issue. For the Image Stream it is best to use the highest possible fluorescence as single-positive for compensation. Have you tried to compensate with beads?
When you say bleed thru, do you mean you can't compensate? Or do you mean that you only see it when you run your samples?
What is your panel design?
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