I have an investigator who is looking at rat primary epithelial cells as well as at a cell line of rat epithelial cells. We have been trying to set up a simple indirect staining protocol for looking at a cytoplasmic antigen using a mouse monoclonal antibody followed by a secondary conjugated antibody. We are using paraformaldehyde and saponin. We have tried 6 different secondary antibodies - 3 were conjugated to FITC and the other 3 to PE - produced in goat or rabbit, purchased from 3 different manufacturers. The cell lines work very well; the antibodies were titered using the cell lines; all of the secondary antibodies react with the monoclonal and all 6 of the secondaries (FITC and PE) show low levels of nonspecific staining when the secondary is added alone or added following an isotype. The problem is in the primary rat epithelial cells - things work fine with each of the 3 PE secondary antibodies but all 3 of the FITC antibodies give huge amounts of nonspecific staining when used by themselves - up in the 4th decade on log scale. Can anybody explain why only the FITC preps and not the PE preps show this high background - and only with primary cells and not the cell line? Thanks in advance -- Barbara J Taylor Manager, FACS Core Lab Division of Basic Sciences NCI, NIH Bldg 37 Room 6008 37 Convent Dr Bethesda, MD 20892 phone 301.594.6892 fax 301.496.8709 taylorba@pop.nci.nih.gov
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