Calman Prussin wrote- >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? If the nonspecific binding indeed results from interactions of antibody with basic sites on eosinophil granule proteins, you probably want a more acidic molecule than poly-glutamic acid as a blocker. Sulfonic acid dyes are better differential stains for eosinophils (i.e., appear to have higher selectivity and binding affinity) than is eosin, which has phenolic and carboxyl groups. If you are not using UV excitation, you might find a dye such as Calcofluor white M2R (a disulfonate) or Uvitex 2B (a tetrasulfonate) useful in blocking the binding. Heparin might also work, although more expensive. -Howard
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