Bs1782

Immunohistochemical measurements of the expression of PI3K, Akt, PTEN and RhoA in the endometrial tissue of the mice in the pregnant group, the pseudopregnant group and the group injected with the PI3K inhibitor were carried out on day 5. The uterus was fixed with 4% paraformaldehyde, dehyderated with graded ethanol, wrapped with xylene transparent paraffin and sliced into sections (5-μm-thick) prior to immunohistochemical staining. Immunohistochemistry was performed using the SP-9001 Reagent kit (Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China). The sections were then stained with primary antibodies to PI3K (1:50; PI3-kinase p110 α antibody, BSA1236; Bioworld Technology, Inc.), Akt [1:80; Akt (P125) antibody, BS3987; Bioworld Technology, Inc.], phosphorylated (p-)Akt [1:80; p-Akt (S473) pAb antibody, BS4006; Bioworld Technology, Inc.], PTEN [1:80; PTEN (D386) antibody, BS5534; Bioworld Technology, Inc.], RhoA [1:50; RhoA (R182) antibody, BS1782; Bioworld Technology, Inc.]. The sections were then examined under a microscope (Olympus BX51) and images were acquired. The intensity of positive expression was determined using Image Pro^®Plus v. 6.0 software. Phosphate-buffered saline (PBS) was used instead of the primary antibody for the negative control.

Standard immunohistochemistry protocols were applied to small coronary arteries treated for 24 h with the various experimental solutions, in order to determine the protein expression levels of RhoA, ROCK1, and ROCK2. The small coronary arteries were fixed with 4 % paraformaldehyde and sectioned (4 μm). The following primary antibodies (100 μL) were used: anti-RhoA (1:200 dilution; BS1782; Bioworld Technology Inc., St. Louis Park, MN, USA), anti-ROCK1 (1:200 dilution; sc-6055; Santa Cruz Biotechnology), and anti-ROCK2 (1:250 dilution; sc-1851; Santa Cruz Biotechnology). The secondary antibody was biotinylated goat anti-rabbit IgG (Beyotime Institute of Biotechnology, Shanghai, China). Sections were visualized with diaminobenzidine (DAB; Shanghai Jierdun Biotech) and counterstained with hematoxylin and eosin (H&E). Images were captured with a digital camera (Nikon) and analyzed using the IMS imaging processing system (Shanghai Jierdun Biotech). Positively stained regions were counted and analyzed. Cardiomyocytes were excluded.