Ab32048

After 4% PFA fixation, cells were blocked by 5% bovine serum albumin (BSA, A7906, Sigma) in DPBS containing 0.1% Triton X-100 for 1 h, followed by the incubation of primary antibodies against integrin beta 1 (12G10, ab30349, Abcam, 1:200), paxillin (Y113, ab32048, Abcam, 1:200), CD49c (integrin alpha 3, ASC-1, MA5-28565, Invitrogen, 1:50), talin 1 (8D4, ab157808, Abcam, 1:100), vinculin (EPR8185, ab129002, Abcam, 1:100), FAK (#3285, Cell signaling Technology, 1:200), α-actinin (H-2, sc-17829, Santa Cruz Biotechnology, 1:200), and Phospho-Myosin Light Chain 2 (Thr18/Ser19) (pMLC, #3674, Cell Signaling Technology, 1:100) in 1% BSA for overnight at 4 °C. The samples were washed twice with PBS before applying fluorescence-conjugated secondary antibodies, including Goat Anti-Mouse lgG H&L (Alexa Fluor® 488) (ab150113, Abcam, 1:1000), Goat Anti-Rabbit lgG H&L (Alexa Fluor® 488) (ab150077, Abcam, 1:1000), Goat Anti-Rabbit lgG H&L (Alexa Fluor® 568) (ab175471, Abcam, 1:1000), Donkey Anti-Rabbit lgG H&L (Alexa Fluor® 647) (ab150075, Abcam, 1:1000), Goat Anti-Mouse lgG H&L (Alexa Fluor® 568) (ab175473, Abcam, 1:1000), Goat Anti-Mouse lgG H&L (Alexa Fluor® 647) (ab150115, Abcam, 1:1000), in 1% BSA with or without dye-conjugated phalloidin and DAPI (R37606, Invitrogen) at room temperature for 45 min.

After treatment with 200 ng/ml CCL7 recombinant protein (#282-P3, R&D, Minneapolis, MN, USA) for 1, 3, 6, and 12 hours, cells were suspended in PBS and incubated with FcR blocking reagent (Miltenyi Biotec, Gladbach, Germany) for 10 min. Cells were stained for 30-40 min at 4°C with the following directly conjugated antibodies: anti-human E-Cadherin-APC (#180224, R&D, Minneapolis, MN, USA), anti-human CCR1 (#PA-141062, Thermo), anti-human CCR2 (#ab32144, Abcam, Cambridge, MA), anti-human CCR3 (#ab32512, Abcam, Cambridge, MA), anti-human CCR5 (#ab32048, Abcam, Cambridge, MA), and anti-human IgG-APC isotype (Miltenyi Biotec, Miltenyi, Germany). Secondary antibody for CCR1, -2, -3, and -5 was conjugated to PE (#12-4739-81, Ebioscience). IgG isotype antibodies were used in parallel as control. Cells were analyzed on an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) with CFlow software (BD Biosciences, San Jose, CA, USA).