Ab138002

A standard protocol was used for immunohistochemical staining. Sections were incubated with primary antibody, matrix metalloproteinase 13 (MMP-13) (ab39012, Abcam, 1:400), type X collagen (Col X, ab58632, Abcam, 1:100), LIF (ab138002, Abcam, 1:200), sclerostin (ab63097, Abcam, 1:50), β-catenin (ab16051, Abcam, 1:200), and osteocalcin (ab93876, Abcam, 1:200), overnight at 4 °C. Subsequently, the polymer detection system (ZSGB BIO) was used to detect the immune activity, and then hematoxylin (ZSGB BIO) was used for counterstaining. For immunofluorescence staining, sections were incubated with Alexa Fluor® 488 goat anti-rabbit secondary antibody (ab150077, Abcam, 1:500) for 1 h at 37 °C in the dark. The morphological measurement of the subchondral bone tissue was performed using a Leica microscope (MD3000), the cartilage was evaluated by Zeiss fluorescence microscope (AXIO OBSERVERA1), and the quantitative analysis was performed blindly. For MMP-13 and Col X, the positive staining number and the total number of chondrocytes in the entire articular cartilage were calculated. For TRAP-positive osteoclasts, LIF, sclerostin, β-catenin, and osteocalcin in subchondral bone, three visual fields of each sample were taken for positive cell count and bone surface length measurement for analysis.

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).

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.