4 well glass slides

Cells were grown on 4 well glass slides (Millipore) in normal growth medium for 24 h. After fixation the cells were, permeabilized using the fixation/permeabilization kit (BD Biosciences catalogue 554714) and incubated with primary mouse anti‐human LAMP2 (Abcam) or EEA‐1 (BD Biosciences) monoclonal antibodies and an Alexa Fluor 488 conjugated goat anti‐mouse IgG (H + L) antibody. The slides were mounted using ProLong Gold antifade Mountant with DAPI nuclear stain (Thermo Fisher Scientific). The slides were examined using a Leica Sp5 confocal microscope and serial 0.3−0.5 µm Z‐stacks images per field (5−7 cells) were acquired. To analyse acidic lysosomes in situ, the cells were incubated with LysoTracker red DND‐99 (Thermo Fisher Scientific) and examined by confocal imaging. Images were analyzed using ImageJ software.

Cells were seeded on 4 well glass slides (Millipore) and infected at an MOI of 1 for 20 h. At various times post-infection, slides were gently washed with PBS and fixed with ice-cold methanol for 20 min at −20 °C. After washing again with PBS, they were blocked at room temperature for 1 h with 3% bovine serum albumin (BSA) diluted in PBS. Slides were then probed with the ORF M antibody at a concentration of 1:100 in 1% BSA in TBS-T overnight at 4 °C. After washing gently with TBS-T 3 times for 15 min each, the slides were incubated at room temperature for 1 h in the dark with Alexa Fluor 488 conjugated anti-Rabbit antibody (#A-11034, Invitrogen, Thermo Fisher Scientific) diluted to 1:100 in 1% BSA in TBS-T. After washing again with PBS 3 times for 15 min each, the slides were mounted using a coverslip and DAPI gel mounting medium (#F6057, Sigma-Aldrich, Darmstadt, Germany) and visualized using a Zeiss Axio Scope.A1 microscope and DAPI and FITC filters.