Alexa fluor 488 conjugated affinipure goat anti rabbit igg h l

Cover slips were treated with poly‐D‐lysine overnight before SACC cells were plated. After different treatments, cells were fixed with 4% paraformaldehyde in PBS for 20 min. Then, cells were permeabilized and blocked with goat serum with 0.3% Triton X‐100 at room temperature for 1 h. Then, samples were incubated with primary antibody (rabbit anti‐γH2AX, Abcam, 1:200) at 4°C overnight followed by one wash and then, incubation with secondary antibody (Alexa Fluor 488‐conjugated Affinipure Goat Anti‐Rabbit IgG(H + L), 1:200, Proteintech, SA00006‐2) for 1 h at room temperature. Cells were stained with DAPI (Solarbio, C0065) for 5–10 min, washed with PBS and then, examined using an Olympus IX71 microscope (Olympus Corporation). For nuclear fluorescence staining, the fixed cells were directly stained by DAPI before imaging.

2.5 × 10⁴ GSMCs in logarithmic phase were planted on a 24-well plate with a chamber slide in each well. Cells were cultured for 12 h, washed with PBS three times and fixed in 4% paraformaldehyde (Leagene, Beijing, China) for 20 min. After washed with PBS (Hyclone, Logan, UT), cells were incubated with 0.5% Triton X-100 (Solarbio, Beijing, China) for 10 min, washed with PBS again, and then blocked with goat serum (Boster, Wuhan, China) for 30 min. Cells were incubated with rabbit anti-mouse smooth muscle actin specific antibody (alpha-SMA, Proteintech, Wuhan, China, lot: 00056560) at dilution of 1:200 at 4 °C overnight. After washed with 0.1% PBST three times, cells were incubated with Alexa Fluor 488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Proteintech, Wuhan, China, lot: 20000098) at dilution of 1:200 in the dark for 1 h, and then stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China). Each chamber slide cell side was placed down on a 100 μL drop of Fluoromount-G™ mounting medium (Yeasen, Shanghai, China) on a coverslip and mounted with nail polish. Cells were observed under a BDS200-FL inverted fluorescence microscope (Optec, Chongqing, China) and recorded with ZOE Fluorescent Cell Imager (Bio-Rad, Hercules, CA).