Hi, Has anyone ever tried to co-stain double pos. cells with one marker in APC, the other in APC-Cy7? We routinely costain murine MHCII and CD11c. Now we need those markers in APC and APCCy7 resp. We use a rat anti-mouse MHCII and a biotinylated hamster anti-mouse CD11c, followed by APC-goat anti-rat (Caltag) and streptavidin-PharRed (Pharmingen). When used separately, both colours are reasonable (although I always found PharRed to be quite disappointing). However when used together on the cells coexpressing classII and CD11c, both signals from those secundary reagents collapse to near oblivion... Could there be steric hindrance? Quenching? Or something stoopid I'm missing? I'd love to hear suggestions or anyone with similar observations! Karim
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