positive negatives in immunofluorescence - (Jan/11/2006 )

hi there,

i am in the process of setting up an immunofluorescence method to detect a cell curface receptor in a rodent cell line.... what i have found so far is that i get almost as intense staining in my negative control [no primary antibody added] as in my sample slides.... any ideas? do i need more washes? is it due to my secondary FITC conjugated antibody?

i use cultured cells which are cytospun down onto polylysine coated slides

primary @ 1:200 dilution (5ug/ml) 1 hour RT
secondary (anti-rabbit IgG FITC conjugated) @ 1:750 1 hour RT
four washes after each in 1%BSA in PBS-T
slides are mounted in vectashield with DAPI

any help is much appreciated

dr bob

ever considered blocking??

before add primary antibody , the slide should be blocked with BSA or normal serum