Gb11034b

Total protein was extracted from endometrium tissues using RIPA buffer (Servicebio, China) supplemented with 1% PMSF (Servicebio, China). Protein quantification was performed using an Enhanced BCA Protein Assay Kit (Servicebio, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out for protein separation. Then, the protein samples were transferred onto the PVDF membrane (Servicebio, China) and blocked in 5% BSA (Servicebio, China). Subsequently, the membranes were incubated with the primary antibodies against HOXA10 (ab191470, Abcam, UK), LIF (ab138002), VEGF (GB11034B, Servicebio, China), VEGFR2 (GB11190, Servicebio, China), P-PI3K (GB11190, Servicebio, China), AKT (GB111114, Servicebio, China), P-AKT (AF3242, Affinity, USA), and ACTIN (GB15001, Servicebio, China) at 4°C overnight. After washing with TBS-T three times, the membranes were then incubated with the secondary antibody at RT for 1h and detected using an ECL plus kit (G2019, Servicebio, China). PhotoShop software (alphaEaseFC, Alpha Innotech, USA) was used to remove the color, and Alpha software (Adobe PhotoShop, Adobe, USA) was used to analyze the optical density of the target band.

Samples were fixed with fresh 4% paraformaldehyde and underwent conventional histological procedures for embedding in paraffin. These samples were cut into 4.5-μm-thick sections, processed for IHC staining with anti-VEGF polyclonal antibody (GB11034B, Servicebio, China) and rabbit anti-Ang-1 polyclonal antibody (ab95230, Abcam, UK), and then incubated with secondary antibodies. The images were digitized by fluorescence microscopy using an IX73 microscope (Olympus Corporation, Japan). The positive cells' density of each slice was measured by image analysis software Image-Pro Plus 6.0.