Immunostaining

Cells were seeded on glass coverslips, fixed with 3% formaldehyde (FA; 18814; Polysciences) for 15 min on ice, and permeabilized or not with 0.05% saponin (S7900; Merck/Sigma) in PBS. For cells grown on unlabelled or Oregon Green–labelled gelatin, 0.1% Triton X-100 (Merck/Sigma) was used instead of saponin for 5 min. Fixed cells were then stained with primary antibodies at room temperature for 1 h, washed in PBS/saponin, stained with fluorescently labelled secondary antibody for 1 h, washed in PBS, and mounted with Mowiol containing 2 µg ml⁻¹ Hoechst 33342 (H3570; Thermo Fisher Scientific). For the detection of endosomal markers, cells were permeabilized for 5 min on ice with 0.05% saponin in PEM buffer [(0.1 M Pipes (P7643, Merck/Sigma), 2 mM EGTA (E3889, Merck/Sigma), and 1 mM MgSO₄ (105886, Merck/Sigma) pH 6.95)] before fixation in order to decrease the fluorescent signal from the cytosolic pool of the proteins⁷⁴. For Airyscan microscopy, nuclei were stained in PBS/Hoechst 33342 (2 µg ml⁻¹) for 10 min and mounted in ProLong Diamond ({"type":"entrez-protein","attrs":{"text":"P36961","term_id":"547831","term_text":"P36961"}}P36961; Thermo Fisher Scientific).