I am using confocal microscopy to confirm the flow cytometry results regarding CD24+ cells. The mammalian cell line is expected to be CD24+. as confirmed by our FACS experiments. However, standard confocal sections are showing a cytoplasmic staining without the expected localization of CD24 at the membrane. The permeabilization has been performed with PBS-T (0.1% Triton in PBS) at RT for 1 min. Anti-CD24 is a ML5 clone available from BD Biosciences. The incubation is performed overnight at 4ºC. Any suggestion to improve the staining protocol used in the microscopy analysis?
I suspect it will improve if you don't use the triton. The triton is a detergent which is used to breakdown the plasma membrane and get antibodies intracellularly. Since you are aiming to see CD24 on the membrane then it is best to leave it intact! You should get minimal intracellular labelling.
TritonX100 permeabilizes cells. Avoid TritonX100 throughout the protocol.
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