Eclipse ti a1r a1 confocal microscope

RPE-1 BRAF V600E cells were grown directly on collagen IV (Sigma) -coated coverslips, fixed in 3.7% formalin, permeabilized using 0.1% Triton X-100, and treated with 0.1% SDS. They were blocked in 1% BSA and then incubated with primary antibody diluted in blocking solution in a humidity chamber at 4°C overnight, washed with 1X PBS, and incubated with secondary antibody. Cells were mounted using mounting media containing DAPI (Vector Laboratories). All secondary antibodies were Alexa Fluor conjugates (488, 555, and 647) (Thermofisher) used at a 1:500 dilution. All images were acquired on a Leica DM 600 inverted microscope at 100X magnification. Z stack images were taken at 0.20µM each.
For melanocyte binucleate counts and immunofluorescence staining, zebrafish adult dorsal scales were fixed using 4% paraformaldehyde for two hours, washed with PBST (PBS+ 0.1% Triton X) and water then blocked in 1%BSA/PBS for 30 minutes prior to primary antibody incubation. Affinity-purified anti-Mitfa antibodies were used at a 1:100 dilution for staining. All images were acquired on a Nikon Eclipse Ti A1R-A1 confocal microscope. Additional information is provided in Extended Experimental Procedures.