Hi, want localize the bovine sperm surface proteins, please anyone share your experience on immunofluorescence staining to locate sperm surface proteins by secondary antibody labelled with FITC?
Dear Somashekar,
for this purpose you need primary antibodies against the surface sperm proteins, the antibodies should be other than bovine (e.g. rabbit, mouse etc.), the secondary antibodies labelled by the FITC should be against the primary immunoglobulin of the selected species. More you need the PBS for the dilution of the antibodies, serum albumin (e.g. human) for the reduction of nonspecific binding of the antibodies, slides suitable for the fluorescent microscopy and the fluorescent microscope.
It is necessary to find suitable primary and secondary antibodies dilution (e.g. 1:10, 1:100). The dilution is made usually by the PBS containing 0.5 per cent of albumin.
The design is: 1. probe – incubation with the primary antibody, washing using PBS with albumin, incubation with the secondary labelled antibody, washing using PBS with albumin, mounting into fresh PBS, observation. 2. control– incubation with the secondary labelled antibody, washing using PBS with albumin, mounting into fresh PBS, observation.
Best regards
Pavel
Dear Dr. Pavel,
Thank you very much for your kind information.
I was following the same protocol with TBST buffer and Blocking with bovine serum albumin (BSA, 3%) and antibody dilution as suggested above, since I am using BSA this might be the reason for fluorescence in negative controls slides?. If possible, kindly provide me the full length protocol for immunofluorescence staining in sperm.
Thanking you
Dear Somashekar,
the protocol strongly depends on the primary antibodies used. Try various dilutions and times. The control positivity may be caused or by the high antibody concentration or by the BSA using. Use another non bovine albumin/protein.
Best regards
Pavel
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