Schiff s reagent

Periodic acid–Schiff (PAS) staining was used to verify the glycocalyx content of the tissue samples. The cryosections were fixed with ice-cold methanol (Honeywell, Offenbach, Germany) for 10 min and the slides were incubated in 1% periodic acid (Carl Roth, Karlsruhe, Germany) for the next 10 min. The acid was washed with running water and the samples were incubated for 10 min with Schiff’s reagent (Carl Roth, Karlsruhe, Germany) and washed under running water again. The slides were placed in a container filled with Harris Hematoxylin Solutions (HHS) (Sigma-Aldrich, Taufkirchen, Germany) for 10 min and differentiated with hydrochloric acid–alcohol solution (AppliChem, Darmstadt, Germany) for 3 s. After that, they were washed and left in the water for 10 min to remove the excess staining. The samples were dehydrated first with 2 times 96% (v/v) ethanol (VWR, Darmstadt, Germany) and then with 2 times 99% (v/v)). The sections were “dipped” into a row of xylene (VWR, Darmstadt, Germany) (four vessels filled with xylene) and embedded with Eukitt (Sigma-Aldrich, Taufkirchen, Germany).

The PAS reaction is used to visualize glycogen-storing cells in the spongiotrophoblast layer and was described previously in detail [15 (link)]. 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).