Pre immune serum

Fresh uterine tissue was fixed in 4% paraformaldehyde, embedded in conventional paraffin, and sliced at 3–8 μm. After dehydration, the sections were placed in citrate buffer and heated to extract antigens. Inactivated endogenous peroxidase was added to the sections in hydrogen peroxide/methanol for 1 h followed by washing in PBS. The sections were incubated in 10% pre-immune serum (Maixin-Biotech, Fuzhou, China) for 30 min at 37 °C prior to incubation with anti-GRP78 antibody (Proteintech) for 12 h at 4 °C. Preimmune serum was used as a negative control. After washing, the sections were incubated with biotinylated antibiotic-rabbit IgG antibody (Maixin-Biotech, Fuzhou, China) for 30 min at 37 °C. Streptavidin labeled with horseradish peroxidase was added with incubation at 37 °C for 30 min. Microscopy (Nikon, Tokyo, Japan) was used to evaluate the sections after staining with DAB chromogenic solution (Maixin-Biotech, Fuzhou, China).

Goat uterine tissues were fixed with 4% paraformaldehyde for 24 h and dehydrated through a graded series of ethanol solutions. Sections (thickness, 7 μm) were mounted onto glass slides precoated with poly-L-lysine solution. The dehydrated sections were placed in citrate buffer (PH 6.0) for antigen retrieval using the microwave heating method. After washing in phosphate-buffered saline (PBS), the slides were placed in 0.3% hydrogen peroxide/methanol for 1 h to inactivate endogenous peroxidase. The sections were then incubated in 10% pre-immune serum (Maixin-Biotech, Fuzhou, China) for 30 min at 37 °C prior to incubation with anti-YPEL3 antibody (1:100, ABclonal Technology, Wuhan, China) for 12 h at 4 °C. The negative control sections were incubated overnight with pre-immune serum. After washing, the sections were incubated with biotinylated anti-rabbit IgG antibody (Maixin-Biotech, Fuzhou, China) for 30 min at 37 °C, washed three times with PBS and then incubated with horseradish-peroxidase-labeled streptavidin for 30 min at 37 °C. Finally, after staining with DAB chromogenic solution, the sections were valuated under a microscope (Nikon, Tokyo, Japan). All antibodies were diluted with PBS.