Sc 179

Uterine horns collected from either miR-1291 antagomir or NC treated mice were frozen in Tissue-Tek^® O.C.T. Compound and sectioned into 9 µm pieces for immunofluorescence staining. Non-specific antigens in the sections were blocked using 10% goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd.) for 1 h following antigen retrieval. Antigen retrieval was performed in a pressure cooker using EDTA (1:50 dilution, pH 9.0) for 20 min in boiling water. Sections were then incubated overnight at 4°C with primary antibodies raised against ArhGAP29 (NBP1-05989; 1:50; Novus Biologicals Canada ULC), RhoA (sc179; 1:100; Santa Cruz Biotechnology, Inc.) and ROCK1 (ab45171; 1:100; Abcam). Subsequently, Alexa Fluor 488 goat anti-rabbit IgG antibody (A11008; 1:200; Thermo Fisher Scientific, Inc.) was used as a secondary antibody and incubated with the sections for 1 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 200 ng/ml) for 10 min at room temperature. Images were captured using an Olympus microscope (IX51; Olympus Corporation, Tokyo, Japan).

The subjects in the current study included 39 patients with IUAs diagnosed by hysteroscope and 28 normal control cases recruited between July 2014 and June 2015 from the Beijing Obstetrics and Gynecology Hospital. Cases selected were all women of reproductive age. The age of the patients with IUAs ranged from 23–40 years old, while ages in normal control group ranged from 27–40. Normal control endometrial tissues were collected from patients who underwent hysteroscopies due to infertility or other factors during the same period. Formalin-fixed, paraffin-embedded endometrial tissues were obtained from the Department of Pathology in Beijing Obstetrics and Gynecology Hospital (Beijing, China) and were originally obtained as pre-therapeutic biopsies. The study was approved by the Capital Medical University Research Ethics Committee (Beijing, China) following the principles of the Helsinki Declaration, and all participants provided informed consent. Immunohistochemistry, using a commercial primary anti-ArhGAP29 antibody (NBP1-05989; Novus Biologicals Canada ULC, Oakville, ON, Canada), RhoA (sc179; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and ROCK1 (ab45171; 1:200; Abcam, Cambridge, MA, USA) were used to investigate the differential expression of ArhGAP29 and RhoA/ROCK1 in severe IUAs compared with normal endometrial tissue.