Dylight 594

The following primary antibody (Ab) was used: mouse polyAb to Amyloid beta (Biolegend, 803002, 1:100). The following secondary Ab was used: goat polyAb to mouse IgG conjugated to DyLight 594 (MultiSciences, 70-GAM5942, 1:200) or Alexa 555 (Invitrogen, A21422, 1:200).

HeLa cells were cultured overnight on glass coverslips and then treated with inhibitors or subjected to transfection. After being washed three times with PBS, the cells were fixed with ‐20°C prechilled methyl alcohol for 10 min and permeabilized with 0.1% Triton X‐100. After blocking with 5% BSA and 3% goat serum in PBS, the cells were incubated with primary antibodies (CD63, Invitrogen; HRS, Abcam; EEA1, abcam; LAMP1, abcam; Flotillin‐1, Abcam) overnight at 4°C in blocking buffer. The following day, after three washes in PBS, the cells were incubated with secondary antibodies (DyLight 488, DyLight 594, MultiSciences) for 30 min at RT, washed in PBS, and then mounted in antifade mounting medium with DAPI. The samples were imaged using an Olympus IX83‐FV3000 confocal microscope (Olympus). Images were analysed with ImageJ software.