Ab150132

Cellular localization of TASK-3 in MDA-MB-231 and MCF-10F cells was analyzed by immunofluorescence as described previously [16 (link)]. Briefly, cells were seeded on coverslips, fixed in 4% paraformaldehyde (PFA)/1× phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin in 1× PBS containing 0.1% Triton X-100 for 30 min. After incubating in blocking buffer, cells were incubated with anti-TASK-3 antibody (1:100; sc-11317, Santa Cruz Biotechnology, USA) overnight at 4 °C. Negative controls were treated in the same way, replacing the primary antibody with 1× PBS. Cells were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ab150132; Abcam, Cambridge, MA, USA) at 1:1000 dilution, for 1 h at room temperature. Finally, DAPI (4′,6-diamidino-2-phenylindole, 0.1 µg/mL for 5 min) was used to stain cell nuclei. Immunofluorescence images were acquired in a fluorescence microscope (Olympus BX53; Center Valley, PA, USA), coupled to a CCD camera. Digital images were acquired using the Q-Capture Pro 7 software (QImagine, Surrey, Canada). Each staining was done in triplicate in 3 independent experiments.

RAW264. 7 or HGF-1 cells seeded in confocal dishes were fixed with 4% paraformaldehyde at 4°C for 15 minutes after washed by PBS 3 times for 20 minutes each time. The cells were then permeabilized with 0. 2% Triton X-100 for 10 minutes and blocked with 2% BSA for 2 hours, and each step was followed by twice PBS washes to remove residual reagents. After the above steps were completed, primary antibodies were added to the cells and incubated at 4°C overnight. The primary antibodies were added as follows: Piezo1 antibody and CD68 were co-incubated to localize Piezo1 on macrophages; CCR7 (1:200;NB100-712; Novus Biologicals, USA) and CD206 (1:200; #24595; Cell Signaling Technology, USA) were co-incubated to identify macrophage polarization; COL-I (1:200;NB600-408; Novus Biologicals, USA) or COL-III (1:200; NB600-594; Novus Biologicals, USA) was incubated to indicate the existence of collagen in HGF-1. Afterwards, the cells were incubated with donkey anti-goat secondary antibody IgG H&L Alexa Fluor 594 (1:200; ab150132; Abcam, UK) and the goat anti-rabbit IgG H&L Alexa Fluor 488 for one hour at room temperature in the dark. After washing away the secondary antibodies with PBS, DAPI was used to stain the nuclei for 10 minutes in the absence of light. Finally, the images were acquired by the confocal laser scanning microscope and quantitatively analyzed by Image J software.