4.8. Staining with Periodic Acid Schiff Reagent (PAS)

The PAS reaction is used to visualize glycogen-storing cells in the spongiotrophoblast layer and was described previously in detail [15]. In short, deparaffinized and rehydrated slides were incubated with 1% periodic acid (Carl Roth, Karlsruhe, Germany) for 10 min, incubated with Schiff’s reagent (Carl Roth, Karlsruhe, Germany) for 20 min, and then probed with sulfite water for 6 min (10% sodium bisulfite solution, 1 M HCl) to reduce the pseudo-PAS reaction. The slides were dehydrated, mounted with xylene mountant, and digitalized as described above. TIFF files were converted to binary images to quantify the PAS-positive area of the spongiotrophoblast (three sections per placenta) with ImageJ (Version 1.53t).