Col3a1 antibody

Lentivirus-stabilized KGN cell lines, which stably overexpressing lnc-GULP1-2:1 (Lv-lnc-GULP1-2:1) and its control (Lv-EGFP), were re-inoculated on glass coverslips pre-coated with poly-lysine and cultured for 24 h in an atmosphere of 5% CO 2 at high humidity. Cells were treated with 4% paraformaldehyde for 20 min, then gently washed with phosphate buffered saline (PBS) for 3×3 min. Next, cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked by using 5% bovine serum albumin (BSA) in TBST for 30 min at room temperature. After blocking, cells were incubated with COL3A1 antibody (1:100, Santa Cruz) or Ki-67 antibody (1:1000, Cell signaling technology, Beverly, USA) at 4℃ overnight and PBS was used as a negative control. Then cells were washed 3 × 3 min with PBS at room temperature and incubated with Alexa Fluor 594-A niPure Goat Anti-Rabbit IgG (1:600, Jackson, Pennsylvania, USA) for 30 min. After washing with PBS, the nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) (5 μg/ml). Then the coverslips were washed again with PBS 4 × 5 min. Finally, the coverslips were mounted with anti-fade Mounting Medium (Beyotime Biotechnology, Shanghai, China), and the image was observed and collected under a laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

Lentivirus-stabilized KGN cell lines, which stably overexpressing lnc-GULP1-2:1 (Lv-lnc-GULP1-2:1) and its control (Lv-EGFP), were re-inoculated on glass coverslips pre-coated with poly-lysine and cultured for 24 h in an atmosphere of 5% CO 2 at high humidity. Cells were treated with 4% paraformaldehyde for 20 min, then gently washed with phosphate buffered saline (PBS) for 3×3 min. Next, cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked by using 5% bovine serum albumin (BSA) in TBST for 30 min at room temperature. After blocking, cells were incubated with COL3A1 antibody (1:100, Santa Cruz) or Ki-67 antibody (1:1000, Cell signaling technology, Beverly, USA) at 4℃ overnight; PBS was used as a negative control. Then cells were washed 3 × 3 min with PBS at room temperature and incubated with Alexa Fluor 594-A niPure Goat Anti-Rabbit IgG (1:600, Jackson, Pennsylvania, USA) for 30 min. After washing with PBS, the nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) (5 μg/ml). Then the coverslips were washed again with PBS 4 × 5 min. Finally, the coverslips were mounted with anti-fade Mounting Medium (Beyotime Biotechnology, Shanghai, China), and the image was observed and collected under a laser scanning microscope (Carl Zeiss, Oberkochen, Germany).