Ab191470

The sections were dewaxed, antigen-retrieved, and blocked. Then, specific antibodies (anti-HOXAs: HOXA1: ab230513; HOXA2: ab229960; HOXA3: ab230879; HOXA10: ab191470, Abcam) covered the sections at 4 °C overnight. After washing three times with PBS, specific secondary antibodies covered the sections for 1 h at 37 °C with horseradish peroxidase, then immersed in diaminobenzidine (DAB) and counterstained with hematoxylin for 2 min. A microscope (BX50/BX-FLA/DP70, OLYMPUS) was used to observe the staining signals. ImageJ Pro (Media Cybernetics, USA) was used by a technician (blinded to the experimental groupings) to quantify the integrated optical densities.

The paraffin-embedded tissues were divided into four groups. The sections were dewaxed to water, antigens were heat-repaired, and endogenous enzymes were inactivated and incubated with the following primary antibodies: cyclooxygenase-2 (COX-2; ab15191, 1:1,000, Abcam, Cambridge, UK), homeobox A10 (HOXA10; ab191470, 1:5,000, Abcam, Cambridge, UK), leukemia inhibitory factor (LIF; ab138002, 1:5,000, Abcam, Cambridge, UK), and Integrin ανβ3 (ab179475, 1:5,000, Abcam, Cambridge, UK) at 4°C overnight. After washing with phosphate-buffered saline (PBS), the secondary antibody was incubated for 30 min, and developed with DBA, then the hematoxylin was restained, and the slices were sealed. The figures were observed and photographed under microscope (BA410T, Motic, Xiamen, China) and analyzed by image processing software (Image-Pro-Plus 6.0, Media Cybernetics, Silver Spring, USA).

Protein was extracted from endometrial tissue samples or cultured cells using RIPA buffer containing protease inhibitors (89,900, Thermo, USA). Equal amounts of 30 μg protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, United States). Then, the membranes were incubated with the following specific primary antibodies: S100P (ab133554, Abcam, UK, 1:1000), Bax (5023 T, Cell Signaling Technology, 1:1000), Bcl-2 (4223S, Cell Signaling Technology, 1:1000), HOXA10 (ab191470, Abcam, UK, 1:1000), GAPDH (5174, Cell Signaling Technology, 1:1000). Afterwards, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime Biotechnology, China, 1:1000). Finally, protein bands were detected by enhanced chemiluminescence (ECL) according to the manufacturer’s (Millipore) instructions.

LAD tissue sections were dewaxed with xylene and hydrated with alcohol at descending concentrations. Following antigen retrieval, the tissues were incubated with goat polyclonal antibody to HOXA10 (1: 200; ab191470; Abcam) at 4 °C overnight, as well as with biotinylated rabbit anti-goat secondary antibody (ab97100; Abcam) at 37 °C for 30 min. Diaminobenzidine (Bost Biotechnology Co., Ltd., Wuhan, China) was added stained for 1–2 min, while hematoxylin (KeyGEN Biotech Co., Ltd., Nanjing, Jiangsu, China) was counterstained for 1 min. The tissue sections were subsequently dehydrated and fixed using neutral balm. Five fields of view were selected and observed under an optical microscope (200 ×, Nikon, Tokyo, Japan) with 100 cells from each field analyzed. Brown-yellow cells were considered to be positive. Cells with a staining degree greater than 25% were considered to be positive cells with the positive rate calculated using the following formula: positive rate = (the number of positive cells/the number of total cells) × 100%.

Total proteins were extracted from mice endometrial tissues. WB was used to detect the expression of proteins COX-2, Integrin ανβ3, LIF, and HOXA10. The protein was adsorbed on the PVDF membrane by gel electrophoresis and sealed with 5% skim milk solution for 2 h at room temperature. The primary antibody was incubated with COX-2 (ab15191, 1:1,000, Abcam, Cambridge, UK), HOXA10 (ab191470, 1:5,000, Abcam, Cambridge, UK), LIF (ab138002, 1:5,000, Abcam, Cambridge, UK), Integrin ανβ3 (ab179475, 1:5,000, Abcam, Cambridge, UK) and β-actin (66009-1-Ig, 1:5,000, Proteintech, USA) overnight at 4°C, washed three times with PBS with Tween (PBST), and secondary antibodies anti-rabbit IgG (#SA00001-2, dilution 1:6,000, Proteintech, Chicago, USA) and anti-mouse IgG (#SA00001-1, dilution 1:5,000, Proteintech, Chicago, USA) were incubated for 1.5 h at room temperature. The PBST was washed three times, and the membrane was incubated with SuperECL Plus (#K-12045-D50, Advansta, Menlo Park, USA) for 1 min. The chemiluminescence imaging system (ChemiScope 6100, Clinx, Shanghai, China) was used for scanning and imaging. β-actin was used as an internal reference for detecting relative expression levels.

Mice uteri were homogenized in RIPA lysis buffer and centrifuged at 10000 xG for 15 min at 4 °C to transfer the supernatant. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after measuring their concentrations and were transferred to a polyvinylidene fluoride membrane. The membrane was blocked with Tris-buffered saline (TBS) containing 5% BSA for 1 h and incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: HOXA10 (ab191470, Abcam), cytokeratin (sc-70926, Santa Cruz), ITGB3 (ab179473, Abcam), LIF (ab138002, Abcam), GAPDH (HRP-60004, Protintech). After washing in TBS three times, membranes were incubated with 1:1000 peroxidase-conjugated goat anti-rabbit or mouse immunoglobulin G (IgG) for 2 h at room temperature.