Hi,

I have a specific antibody conjugated alexa fluor 488 to stain tight junction proteins. The free space lifetime is around 4. Based on protocol, I kept cells with stain for an overnight at 4 degree. The images are good with confocal, however, the fluorescent lifetime imaging shows very small lifetimes which is homogenously distributed every where (in the range of 0.2-0.7). I am afraid an overnight keeping of cells with antibody conjugated stains may cause fluorescence quenching due to the increase of the stain conjugated molecules concentration inside the cells. Has anybody encounters such problem? Do you have any idea how long would be the best immunostaining duration to have enough intensity and proper lifetime?