Placental Calcification Quantification

The von Kossa staining was performed as previously described in Speer et al. 2009 [31 (link)] with the inclusion of an optimally reduced 22 min von Kossa treatment. Placental tissue section from 8 normotensive pregnant women and 8 preeclamptic women were stained, and images of mounted sections were acquired on a Keyence BZ-X800 microscope using the Keyence BZ-X software (Keyence, Ozaka, Japan) at 4× magnification. File names were coded. Individual calcified lesions were imaged on a Nikon E800 Upright Microscope (Nikon Corp., Tokyo, Japan) at 2.5 and 10× magnification and file names were coded. Semi-quantitative analysis of calcified lesions was performed on all tissue sections using ImageJ/Fiji (NIH, Bethesda, MD, USA) histomorphometry software.