Goat anti rabbit dylight 594

After 30 days of differentiation, PreMel were seeded in glass coverslips and after 24 h were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature. The cells were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 1% bovine serum albumin (BSA) for 30 min. The cells were incubated with respective antibodies: mouse anti-human PMEL (HMB45 clone, #cat M0634, DAKO, RRID:AB_2335682), rabbit anti-MITF (cat# 122982, Abcam, RRID:AB_10902226), or mouse anti-tyrosinase (#cat sc20035, Santa Cruz, RRID:AB_628420) at 4 °C overnight. After the incubation, the cells were washed in PBS and stained with the secondary antibodies: goat anti-mouse DyLight® 488 (35502, Thermo Fisher) or goat anti-rabbit DyLight® 594 (35561, Thermo Fisher). 4′, 6-Diamidino-2-phenylindole (DAPI, Sigma Aldrich) was used for nuclear staining. Coverslips were mounted in FluorSave™ (Merck Millipore, MO, USA) prior to fluorescence microscopy analysis (ECLIPSE TE2000U, Nikon).

Throughout this work, cells were treated with TNF-α (300-01A, PrepoTech, USA) or mitomycin C (MMC, M4287, sigma-aldrich, USA). For immunostaining, we used the nuclear dye Hoechst 33342 (10 μg/mL; H1399, Thermo Scientific, USA) and cytoplasmic dye HCS CellMask Deep Red (5 μg/mL, H10294, Thermo Scientific, USA), primary antibodies: Anti-N-Cadherin mouse IgG2ab mAb (SC-271386, Santa Cruz Biotechnology, USA), rabbit anti-snail/Slug (ab180714, abcam, USA), Goat anti-Vimentin (sc-7558, Santa Cruz Biotechnology) and Mouse-anti-Caspase-7 (cleaved caspase-7 p10, clone h207, sc-22179, Santa Cruz Biotechnology, USA), as well as secondary antibodies: Goat anti-Rabbit DyLight 488 (dy488, 35553, Thermo Scientific, USA), Goat anti-Rabbit DyLight 594 (35561, Thermo Scientific, USA) DyLight 488 mouse (35503, Thermo Scientific, USA) and Donkey anti-Goat DyLight 594 (SA5-1088, Thermo Scientific, USA). For western blot, we used the antibodies rabbit anti-snail (#3879, Cell Signaling, USA), rabbit anti-slug (#9585, Cell Signaling, USA), rabbit anti-vimentin (#5741, Cell Signaling, USA), rabbit anti N-cadherin (#13116, Cell Signaling, USA), rabbit anti-β-catenin (#8480, Cell Signaling, USA) and mouse anti-β-actin (sc-81178, Santa Cruz, CA).