Dylight 488

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.

Cells were fixed in prewarmed 4% paraformaldehyde for 30 min and permeabilized with 0.2% Triton X-100 for 10 min. After the samples were blocked with 5% BSA, cells were incubated overnight with antibody (1:50 in PBS containing 5% BSA). Staining was detected using DyLight 488- or DyLight 549-labeled secondary antibody (Multiscience). Nuclei were co-stained with DAPI (Roche). Stained cells were imaged using a confocal fluorescence microscope (IX81-FV1000; Olympus). The percentage of L. monocytogenes or Flag-SNX10 that co-localized with Rab5, Rab7 or LAMP1 was quantitatively analyzed using the co-localization dialogue of Metamorph offline software by randomly scanning > 20 cells in each test group in two or more independent experiments [38 (link)].