Water 1.2 na objective

Sixteen hours after transfection, HEK‐293 cells were fixed in 4% v/v paraformaldehyde (PFA) for 20 min and RAJI cells were adhered to poly‐l‐lysine‐coated coverslips and subsequently fixed. Cells were blocked in 3% w/v BSA, 1% w/v filtered human serum, and 10 mm glycine in PBS for 1 h and subsequently stained for 30 min in the same solution supplemented with 10 µg·mL⁻¹ of the following primary antibodies: anti‐CD9 (MEM61; Thermo Fisher Scientific) or anti‐EWI‐F (polyclonal; Thermo Fisher Scientific). This was followed by a 30‐min incubation with an appropriate secondary antibody: anti‐mouse IgG1‐Alexa Fluor 647 or anti‐rabbit IgG‐Alexa Fluor 647 (both Thermo Fisher Scientific). Samples were then stained with DAPI, washed, and embedded in mowiol. For intracellular staining of EWI‐F, cells were permeabilized for 10 min in 0.1% Triton X‐100 in PBS and all subsequent solutions contained 0.5% w/v saponin. Samples were imaged on a SP8 confocal microscope with a 60× water 1.2 NA objective (Leica, Wetzlar, Germany) using appropriate laser lines and settings. Colocalization was analyzed using the Coloc2 application of FIJI, and the other image analysis was performed using cellprofiler [92].