Alexa fluor conjugated anti rabbit

Cells were seeded onto acid-washed coverslips and cultured for at least 24 h before fixation and staining. Cells were fixed (4% paraformaldehyde in PBS for 10’), permeabilized for 1–2’ at room temperature with 0.2% Triton X-100 in KB buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1% bovine serum albumin (BSA)), washed in KB, and blocked with 3% BSA in PBS for 1 h. Primary antibodies anti-5caC (Active Motif), anti-tyrosinase, and anti-Melan-A (both from Santa Cruz Biotechnology) were incubated overnight at 4 °C. After washing twice in KB, samples were incubated with Alexa Fluor-conjugated anti-rabbit (Molecular Probes) for 30’. Nuclei were counterstained with DAPI. The nuclear envelope was detected by immunofluorescence staining of lamin B using a mouse monoclonal antibody (Abcam) and following protocol provided by the vendor. Images were taken using a Leica Microsystems TCS-SP8A confocal microscope controlled by the LAS software. Exposure times were optimized for control samples and identical exposure times were used for experimental samples.