Ab3743

When cells adhered to 50–60%, the culture was terminated. Cells were fixed in 4% paraformaldehyde for 30 min and permeabilized with Triton X-100 (0.3%, ZSGB-BIO, China) for 10 min. Uterine leiomyoma cells were suspended in 10% normal goat serum (Gibco Life Technologies, USA) and then incubated with primary ER-a antibody (1:1,000, Abcam Cat# ab3743, RRID:AB_304042) overnight at 4 °C. Then, cells were washed and incubated in the secondary antibody (1:7,000, Abcam Cat# ab6721, RRID:AB_955447) for 1 h at room temperature. Uterine leiomyoma cells were applied in DAB Kit (ZLI-9017, ZSGB-BIO, China) following the manufacturer’s protocol. When the chromogenic process was completed, we took a photograph under the microscope and made a statistical analysis.

Cells were seeded in 6-well plates and maintained for 48 h. After being washed 3 times with PBS, cells were fixed in 4% paraformaldehyde solution for 30 min, permeabilized with 0.3% Triton X-100 for 10 min, and suspended in 10% normal goat serum (Gibco Life Technologies, USA) for 10 min, then incubated with the primary antibodies: α-Actin (1:1,000, Abcam Cat# ab69512, RRID:AB_2222725), ER-α (1:1,000, Abcam Cat# ab3743, RRID:AB_304042), and ER-β (1:1,000, Abcam Cat# ab3576, RRID:AB_303922) overnight at 4 °C. After another three washes with PBS, the cells were incubated with secondary antibody for 1 h at room temperature. DAPI (4',6-diamidino-2-phenylindole) reagent (Shanghai Leuven Biotechnology Co., Ltd., China) was used to counterstain the nuclei. The result of the staining was observed with an inverted fluorescence microscope system (OLYMPUS, X53, Olympus Corporation, Japan).