Gb3 cd77

For the immunofluorescence analysis, the cells were fixed using 10% of neutral buffered formalin (Sigma-Aldrich #HT5011, St. Louis, MO, USA) at room temperature for 15 min. After the fixation, the samples were permeabilized for 15 min with PBS containing 0.2% of Triton X-100 and subsequently blocked for 1 h with 5% of BSA in PBST (PBS + 0.1% Tween 20). Following this, the samples were subjected to an overnight incubation at 4 °C in a humid chamber with a primary antibody specific to CD31 (dilution 1:50, DAKO, #M0823, or 1:50, Abcam #ab28364), diluted in antibody diluent (Dako, S3022). GB3/CD77 (1:50, Biolegend, San Diego, CA, USA, clone 5B5) was used for co-staining. On the following day, the samples underwent three washes (5 min each) with PBST and were then incubated at room temperature in a humid, dark chamber for one hour with the appropriate secondary antibody (Thermo Fisher, Waltham, MA, USA; Goat anti-mouse-Alexa Fluor 647 or goat anti-rabbit-Alexa Fluor 488) and diluted 1:500 in the antibody diluent. Following the secondary antibody incubation, the samples were treated with Hoechst (dilution 1:5000) for 15 min at room temperature and visualized either through a fluorescence microscopy (Olympus IX81 light microscope) or a confocal microscopy (Zeiss LSM 800, Oberkochen, Germany).

There were 400 nm of latex nanoparticles labeled in red (Abcam # ab269893), which were coated with the Shiga toxin non-toxic subunit B-STxB (Merck Life Science # SML0562, Darmstadt, Germany) following the manufacturer’s instructions. Briefly, the STxB stock was diluted to 0.1 mg/mL with the Reaction Buffer A and mixed with the latex nanoparticles for 15 min. After stopping the reaction, 1 μg of functionalized nanoparticles was added to the cells in a well of the chamber slide cultured for one week and incubated overnight. Then, the immunofluorescence was performed as indicated above using the GB3/CD77 (1:50, Biolegend, clone 5B5) as the primary antibody and goat anti-mouse-Alexa Fluor 488 (1:500, Thermo Fisher) as the secondary antibody.