Mouse anti lamp1

HeLa cells were seeded onto 6-well plates containing sterile coverslips at a density of 7 × 10⁴ cell/mL. Following infections with V. parahaemolyticus strains at an MOI of 10 and addition of CNF1/CNF1 C866A as detailed above, the cells were washed with PBS and fixed in 3.2% (vol/vol) paraformaldehyde for 10 min at room temperature. Fixed cells were washed in PBS and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Nuclei and actin cytoskeleton were stained with Hoechst 33342 (Sigma) and rhodamine-phalloidin (Molecular Probes), respectively, for infection analyses and quantification. For evaluating endosomal localization of bacteria, nuclei, actin cytoskeleton, and LAMP-1 were stained with Hoechst 33342 (Sigma), Alexa Fluor 680-phalloidin (Molecular Probes), and mouse anti-LAMP-1 (Abcam Ab25630), respectively, as described previously (31 (link)). The images were collected using a Zeiss LSM 710 confocal microscope.

The proteolytic degradation of lysosomes was assessed with DQ-ovalbumin (Invitrogen) according to the manufacturer’s instructions. DQ-ovalbumin can form green fluorescent puncta once degraded by lysosomes. In brief, after seeding in sterile culture dishes, each group of treated podocytes was incubated with 4 µg/mL DQ-ovalbumin at 37 °C for 2 h. The podocytes were fixed with 4% paraformaldehyde for 10 min and visualized using a TCS SP5 II confocal microscope. The mean fluorescent intensity of the green-fluorescent DQ-ovalbumin puncta in individual podocytes was calculated and presented as a histogram. For flow cytometric detection, after incubation with 2 µg/mL DQ-ovalbumin for 2 h, the podocytes were trypsinized and re-suspended in PBS. The fluorescent intensity value of DQ-ovalbumin was quantified by flow cytometry.
To determine whether the dequenched DQ-ovalbumin vesicles were co-localized with lysosomes, after DQ-ovalbumin incubation, the cells were fixed with 4% paraformaldehyde at room temperature and permeabilized with ice-cold methanol at −20 °C for 10 min, respectively. Following washes in PBS, the cells were labeled with mouse anti-LAMP1 (Abcam) and visualized by incubation with the secondary antibody. DAPI was used to stain the nuclei. Images were obtained by a TCS SP5 II confocal microscope.