Cell permeabilization affects detection of a surface antigen. (A) Surface csA-SA was detected by incubating unpermeabilized fixed cells with an anti-csA antibody and a fluorescent secondary antibody. (B) Fixed cells were permeabilized with methanol at −20°C and then incubated sequentially with the anti-csA antibody and secondary antibodies. Surface labeling was almost entirely lost when following this classical staining procedure. (C) The surface of fixed cells was labeled prior to permeabilization, then cells were permeabilized and intracellular antigen stained. This two-step procedure allowed the simultaneous labeling of both surface and intracellular antigens. All the pictures presented in this figure were taken sequentially with the same microscope and with identical settings. Scale bar: 5 μm.