Kiss1

The study received institutional approval (#2020-KY-164) and was carried out in accordance with the guidelines from the Zhengzhou University Research Ethics Board. Paraffin-embedded sections (5 μm) obtained from 3 control and 3 PE patients were deparaffinized and rehydrated. Antigen retrieval was conducted by boiling sections in sodium citrate buffer (pH 6.0) for 8 min. Endogenous peroxidase activity was blocked by incubating sections of 3% hydrogen peroxide solution at room temperature for 10 min. After 1 h of blocking with 3% bovine serum albumin in phosphate-buffered saline (PBS), sections were incubated with specific primary antibody overnight at 4 °C. Following primary antibody incubation (KISS1, 1:50, Santa Cruz Biotechnology #sc-101246; ID3, 1:50, OriGene #CF500714), the sections were incubated with HRP-conjugated secondary antibody. Sections were developed using the Peroxidase/DAB Dako REAL EnVision Detection System (Dako) and counterstained with hematoxylin. Negative control in the absence of primary antibody was performed in parallel.

All tissues were fixed in 10% buffered formalin solution and then embedded in paraffin. All tissues were then cut into 4-μm-thick sections. Immunostaining was conducted using the Elivision™ Plus method, and the procedure was performed in accordance with the kit instructions. Samples were deparaffinized using routine methods and dehydrated using xylene and alcohol. Methanol containing 3% H₂O₂ solution was used for blocking endogenous peroxidase activity, and citrate buffer was used to repair antigen. Goat serum was used for blocking. MACC1 (rabbit polyclonal antibody, Santa Cruz Biotechnology, US), CD44 (mouse monoclonal antibody, Abcam, US), Twist1 (mouse monoclonal antibody, Abcam, US), and KiSS-1 (mouse monoclonal antibody, Santa Cruz Biotechnology, US) primary antibodies were added, and then all sections were incubated overnight at 4 °C. Then enhancer (reagent A) and reagent B were added. The images were allowed to develop in diaminobenzidine (DAB) substrate. Finally, all sections were re-dyed with hematoxylin and mounted with gum.