We are performing intracellular staining for nuclear antigens in eosinophils and are getting a large amount of non-specific binding. We have addressed autofluorescence by using APC as the fluorophore- that seems to work. We are using the typical aldehyde fixation (FACSLyse), followed by 80% methanol and then 0.5% saponin in the staining buffer. We have used a variety of techniques to decrease non-specific binding, including: variety of fixation protocols (with & w/o MeOH), titration of the primary and secondary Abs, using both mAb and rabbit pAb primary Abs, blocking with goat serum (species of secondary Ab), various detergents (triton, tween, OGP, NP-40) and probably other maneuvers I can't recall. We get good results and low non-specific binding with monocytes, but the eosinophils continue to evade us. Am next looking to blocking with human IgG (suggested concentration?) or perhaps poly-Glutamic acid (to block all of the basic eosinophil granule proteins). 1. Thoughts on other ways to block non-specific binding in eosinophils? 2. Thoughts on the use of poly-glutamic acid? Thanks, Calman
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