Visualizing SNX1 in HeLa Cells

HeLa cells were plated onto coverslips in 6-well plates (Gibco) at a density of 20,000 cells per well and transfected with siRNA after 24 hours. Cells were incubated for a further 120 hours after transfection before fixation with 4% formaldehyde. Cells were labelled for SNX1 with 1:200 α-SNX1 (611482—BD Transduction Laboratories) antibody and Alexafluor 488 (Thermo Fisher Scientific) secondary antibody as described previously [12 (link)], with an additional 30 minute incubation in 1:10,000 whole cell stain (HCS cell mask red, Thermo Fisher Scientific) before imaging on an AxioImager Z2 widefield fluorescent microscope. 1024 x 1024 images were taken with a 63x lens, with 20 ms exposure for the red channel (cell mask) and 400–800 ms exposure for the SNX1. Exposure times for these markers were constant in individual experimental biological repeats, but were adjusted between experiments so that the imaged pixels were as bright as possible without becoming saturated.