Ultrastructural Analysis of Melanocytes

Control and Rab4A-knockdown melanocytes were seeded on Matrigel coated glass coverslips. Post 24 h, cells were fixed initially with 0.5% Karnovsky’s fixative (4% paraformaldehyde, 72 mM sodium cacodylate pH 7.4, 4 mM CaCl₂, 0.5% glutaraldehyde) for 2 h followed by overnight fixation with 2% Karnovsky’s fixative (contains 2% glutaraldehyde). Cells were processed for Epon embedding as described (Raposo et al., 2001 (link)). Ultrathin sections of cell monolayers were prepared with a Reichert UltracutS ultramicrotome (Leica Microsystems) and contrasted with uranyl acetate and lead citrate as described (Raposo et al., 2001 (link)). Samples were examined with a FEI Tecnai Spirit electron microscope (FEI Company), and digital acquisitions were made with a numeric camera (Quemesa; Soft Imaging System). For quantification, melanosome stages were defined by morphology (Raposo et al., 2001 (link)) and vacuoles were defined as empty organelles. Melanosomes and vacuoles per µm2 cytosol were counted using ImageJ software. We have counted 10 cells each from control sh and Rab4A sh condition. Further, we have estimated the melanosome stages from 883 total melanosomes of control sh and 300 total melanosomes of Rab4A sh cells.