Ab72591

Formalin-fixed, paraffin-embedded uteri were subjected to routine 5 µm thickness sectioning for IHC analysis, and all IHC experiments were performed using a standard protocol. Briefly, sections were deparaffinized by incubating with xylene for 30 mins and rehydrated in an ethanol series, followed by antigen retrieval and endogenous peroxidase blockage. Sections were then incubated with respective primary antibodies, including anti-LIF (1:50; DF13730; Affinity), integrin β3(1:50; AF6086; Affinity), HoxA10 (1:50; #58891; Cell Signaling Technology), and HoxA11 (1:50; ab72591; Abcam), overnight at 4 ℃ in a humidified chamber, followed by incubation with the corresponding secondary antibodies. Phosphate-buffered saline was used as the negative control instead of the primary antibody. Image Pro-Plus software (Iv.6.0; Media Cybernetics) was used to evaluate the reactivity of the endometrial glands and luminal surface epithelium of the uterus (average positively-stained area percentage) using two independent analyzers.

Transfected cells were fixed with 4% formaldehyde, washed by PBS, treated with 0.5% Triton X-100, and then cultured at 4 °C for 5 min. Next, the cells were treated with a HOXA11-AS FISH probe (RiboBio; Guangzhou, China) mixed in 100 μL of hybridization buffer at 37 °C overnight. After hybridization, the slides were washed and treated with 4′6-diamidine 2-phenylindole (DAPI; Shanghai Beyotime Biotechnology Co., Ltd., China).
IF assays were conducted in a manner similar to FISH assays. Briefly, the cells were fixed with 4% formaldehyde, blocked with 5% BSA, and then incubated with anti-HOXA11 antibody (#ab72591, Abcam; 1:800), followed by incubation with a goat anti-mouse fluorescent secondary antibody (#ab150115, Abcam; 1:500) for 48 h. The cells were then stained with DAPI and observed under a fluorescence microscope (Leica, Germany). Five randomly selected visual fields in each group were observed, photographed, and analyzed for staining.