Immunocytochemistry of Paraformaldehyde-fixed Cells

Immunocytochemistry was carried out on paraformaldehyde-fixed cells grown on glass cover slips as described earlier [4] (link). In brief, cells were permeabilized and blocked with 3% BSA and 0.1% saponin (both Roth, Karlsruhe, Germany) in PBS, stained with the primary antibodies diluted in blocking buffer for 1 h at room temperature or overnight at 4°C following incubation with the secondary antibody diluted in blocking buffer for 30 min at room temperature. Between all steps cells were triple washed with 0.1% saponin in PBS and then mounted on glass microscopic slides in ProLong Gold anti-fade reagent (Invitrogen Life Technology). The images were taken with an Axioplan II fluorescence microscope and AxioCam MRm camera and processed by Axiovision 4.7 (all Zeiss, Oberkochen, Germany). All samples were counted blind.