Anti phalloidin alexa488

EC were used for PLA in situ detection using the Duolink Detection Kit (Olin Bioscience, Uppsala, Sweden) according to the manufacturer’s protocol. Briefly, Cells grown on coverslips were starved for 3 h and treated with HGF or S1P for 5 min, then washed with cold PBS and fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton and blocked with 1X blocking solution. Fixed cells were then incubated overnight with anti-ITGB4 and anti-S1PR3 or anti-S1PR2, or incubated with anti-S1PR1and anti-S1PR2 antibodies at 4 °C. Proximity ligation was performed with PLA PLUS and MINUS Probes for goat and rabbit. Dapi staining was included in the Duolink Detection Kit while anti-Phalloidin Alexa488 (Invitrogen, Carlsbad, CA) at 1:200 was added during the detection reaction. Lastly, slices were mounted and checked with an epi-fluorescence microscope under a 60x oil objective. Texas-red signal was analyzed via BlobFinder Imaging Software, developed and optimized for the analysis of images generated by the in situ PLA (Uppsala Science Park, Sweden) (Ephstein et al., 2013 (link)). Four fields were randomly chosen for analysis and averaged and three separate samples were examined per condition (approximately 60–80 cells total/condition).

Proximity ligation was performed according to the manufacturer’s protocol using the Duolink Detection Kit with PLA PLUS and MINUS Probes for mouse and rabbit (Olink Bioscience, Uppsala, Sweden). In brief, an oligonucleotide-conjugated probe (used as secondary antibody) is directed against primary antibodies raised against gVPLA₂ or LC3. HPAEC grown on glass slides were incubated with chloroquine and/or gVPLA₂ human recombinant protein and then serum starved for 18 hours. Cells were washed with chilled PBS and fixed with 4% paraformaldehyde for 15 min, blocked, and permeabilized with 0.2% Triton-X100 containing 3% BSA, and incubated overnight with appropriate antibodies. DAPI stain was included in the Duolink Detection Kit, while anti-Phalloidin Alexa488 at 1:5000 (Invitrogen) was added during the detection reaction. Specimens were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA) and examined with an epi-fluorescence microscope under a ×60 oil objective. Texas-Red signal was analyzed via BlobFinder Imaging Software, developed and optimized for the analysis of images generated by the in situ PLA (Uppsala Science Park, Sweden). Five fields were randomly chosen for analysis and averaged, and four separate samples were examined per condition (approximately 60 cells).