Application suite x

Cells were seeded onto multispot microscope slides (Hendley-Essex) in culture medium in a humidified incubator at 37°C with 5% CO₂. 24 h after seeding, cells were washed twice in PBS and fixed in 4% PFA in PBS for 20 min at room temperature. Cells were washed twice in PBS and permeabilized for 10 min in 10% Triton X-100. Cells were washed five times in PBS and blocked for 1 h in blocking buffer (20% FBS, 1% Tween-20 in PBS). Blocking buffer was aspirated, and cells were incubated in the primary antibody cocktail diluted in blocking buffer for 1 h. Cells were washed twice in PBS, incubated in blocking buffer for 10 min, and washed twice again in PBS before incubation for 1 h in blocking buffer containing fluorescent secondary antibodies (Data S5). Cells were washed twice in PBS, incubated in blocking buffer for 10 min, and washed twice again. PBS was aspirated, and Vectashield mounting medium (Vector Biolabs) was added to cells before application of the coverslip. The coverslip was sealed with nail varnish, and slides were imaged at room temperature using a 63×/1.4 oil-immersion objective on a Leica TCS SP8 confocal microscope controlled with Leica Application Suite X. Images were normalized without altering gamma using Adobe Photoshop CC 2017.