Alkaline Phosphatase Activity Assay.

Uteri from the artificial decidualization experiment were fixed overnight in 10% neutral buffered formalin followed by incubation in a sucrose gradient and were embedded in optimum cutting temperature (OCT) medium using an ethanol/dry ice bath. Frozen sections (5 µm thickness) were postfixed in 0.2% glutaraldehyde for 10 min, washed in 0.1 M Tris⋅HCl (pH 9.5), and then incubated in 4-nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) working solution (Vector Laboratories) for 15–25 min. Sections were counterstained in Nuclear Fast Red (Vector Laboratories) for 30 s, rinsed in tap water, dehydrated, and then mounted with Permount. A dark blue stain is indicative of alkaline phosphatase activity in the decidualized cells of the uterine stroma.