A11525

Slides were heated in an oven for 2 hours at 60°C, dewaxed, distilled with water for 2 minutes, and followed by high pressure steam distillation. After blocking with a peroxidase in 3% H₂O₂ solution for 10 minutes, washing 3 times with phosphate-buffered saline (PBS) for 5 minutes, the mixture was supplemented with a primary antibody and incubated at 37°C for 60 minutes. After PBS washing in the same manner, secondary antibodies to HMGCS2 (Elabscience, E-AB-13296) and CD31 (Abclonal, A11525), respectively, were added. After incubation at 37°C for 60 minutes in PBS, samples were washed 3 times with PBS for 5 minutes. Each section was colored by diaminobenzidine for 1 to 2 minutes. Each slice was photographed microscopically, and the images were analyzed by IPP6.0 image analysis software.

The ovarian tissue slices as above mentioned was utilized to immunohistochemistry examination. Sections were incubated with primary antibodies against α-SMA (55135-1-AP, Proteintech, Wuhan, China) or CD31 (A11525, Abclonal, Wuhan, China) overnight at 4°C. After washing in PBS, slides stained with α-SMA were incubated with goat against rabbit biotin-labeled antibody (A0277, Beyotime, Shanghai, China) in PBS solution for 60 min at 37°C and conjugated with HRP-labeled Streptavidin (A0303, Beyotime) for 30 min at room temperature. The HRP-labeled goat anti-rabbit antibody (#31460, Thermo Fisher Scientific) was prepared to conjugate with CD31. Then slides were colorated using DAB reagent and counterstained with hematoxylin. The images were shot at × 100 or ×400 magnification.