Cytoperm wash solution

Cells were plated on glass coverslips and, after an additional 48 h, were used for immunofluorescence detection, as described previously (17 (link)). Cells were fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences), washed with BD Cytoperm/wash solution (BD Biosciences), and incubated with the primary antibody in wash-solution (5 µg/ml) at 4°C for 16 h. After washing with wash-solution, the cells were treated with goat anti-mouse IgGAM-rhodamine (Abcam, cat. no. ab6004) or anti-rabbit IgG-FITC (Abcam, cat. no. ab6798) in wash-solution (1:1,000 dilution) at 25°C for 1 h, followed by mounting on a DAPI-containing mounting medium (Vector Laboratories, cat. no. H-1200). Confocal microscopic analysis was performed using a Zeiss LSM 510 Meta microscope (Zeiss AG). The staining with an anti-FBXO2 antibody (mouse IgM; Santa Cruz Biotechnology, cat. no. sc-393873) was performed under the conditions described above to confirm the accessibility of the IgM antibody to the nucleus.