Anti estrogen receptor α

Paraffin sections were dewaxed in xylene, placed in gradient concentrations of ethanol to recover the antigen, and then blocked with goat serum at room temperature for 20 min. Subsequently, the sections were incubated with a primary antibody (anti-estrogen receptor α, Santa Cruz Biotechnology, 1:50; anti-progesterone receptor A, Proteintech, 1:300; anti-VEGF, Santa Cruz, 1:50; anti-FGF-2, Immunoway, 1:100; anti-LIF, Gentex, 1:200; ITG β-3, Abclonal, 1:150) at 4°C overnight, rinsed with phosphate-buffered saline with Tween (PBST) five times for 5 min, and incubated with secondary antibodies (anti-estrogen receptor α, Santa Cruz Biotechnology, 1:50; anti-progesterone receptor A, Proteintech, 1:300; anti-VEGF, Proteintech, 1:50; anti-FGF-2, Bioswamp, 1:100; anti-LIF, Gentex, 1:200; ITG β-3, Absin, 1:150) at room temperature for 1 h. After washing with PBST five times for 5 min, the sections were developed using 3,3’-diaminobenzidine and stabilized using hematoxylin for approximately 3 min. Finally, the sections were dehydrated, made transparent, and sealed. The images were scanned using a nanozoomer slide scanner (Hamamastu, Japan) and observed using the NDP view2 system.

Pregnant mare serum gonadotropin (PMSG) was purchased from the Hangzhou Animal Medicine Factory, China. Human chorionic gonadotrophin (HCG) was provided by Livzon Pharmaceutical Factory, Zhuhai, China. Other materials included anti-vascular endothelial growth factor (VEGF) (Santa Cruz, SC-7269), anti-fibroblast growth factor 2 (FGF-2) (Immunoway, Cat NO.YT5549), anti-estrogen receptor α (Santa Cruz, SC-787); anti-progesterone receptor A (Proteintech, Cat NO.25871-1-AP); anti-LIF, (Gentex, Cat NO.GTX11940), ITG β-3(Abclonal, Cat NO.A2542) and Evans Blue (Cat. number E8010). Main reagents and devices included quantitative real-time PCR (qRT-PCR) equipment (Applied Biosystems, USA), SYBR Green qPCR Kit (Yesen, Cat NO.11201-11203), nucleic acid protein analyzer (DU730, Beckman Coulter, USA), Mastercycler gradient PCR apparatus (Eppendorf, Germany), Nikon microimaging system (TE2000-U, Tokyo, Japan), Step-One Real-Time PCR (Applied Biosystems, California, USA), scanning electron microscope (HITACHI, SU8100, Japan), estrogen ELISA kits (No.501890, Cayman, USA), progesterone ELISA kits (Cat: ELK7894, ELK Biotechnology, China), and Odyssey infrared imaging system (licor Biosciences, USA).

Endometrial tissues were homogenized and lysed in tissue protein extraction reagent, supplemented with a protease inhibitor cocktail, placed on ice for 30 min, and centrifuged at 4°C (12,000 rpm for 10 min). After the protein concentration was determined, the samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and transferred to a PVDF membrane. The membranes were sealed with 5% skim milk at room temperature for 0.5 h, and then incubated with a primary antibody at 4 °C for 24–48 h. Antibodies include anti-estrogen receptor α (Santa Cruz Biotechnology, USA, 1:50); anti-progesterone receptor A (Proteintech, China, 1:200; anti-VEGF, Santa Cruz, China, 1:200); anti-FGF-2 (Immunoway, China, 1:500); anti-LIF (Gentex, China, 1:300); ITGβ3 (Abclonal, China, 1:500); and anti-β-actin (Proteintech, China, 1:1500). PVDF membranes were incubated with fluorescent secondary antibodies (CST, USA) on a shaking table at room temperature for 1 h. Finally, the bands were scanned using Odyssey infrared imaging system (licor Biosciences, USA).