Histosol

Endogenous PIF was detected in placental tissue using immunofluorescence. Briefly, placental samples were fixed in formalin and processed through embedded paraffin for the histological evaluation. Paraffin sections (3 μm) were dewaxed in Histosol (Sigma Chemical Co; St Louis, MO) and rehydrated through descending grade of alcohol (95–70%) to distilled water (dH2O). PIF was detected with a Biotin conjugated antibody against PIF, (Biosynthesis Code: AB1473-1) dil. 1:100 and a secondary reagent Alexa Fluor^® 633 streptavidin (biotin-binding protein) (Thermo Fisher Scientific Code: S-21375, Rockford IL. USA) dil. 1: 200 and counterstained using hematoxylin and eosin (H&E). Finally, samples were mounted with EMS Shield Mount (Electron Microscopy Sciences Code: 17985–150). All images were obtained with a DM6000 B microscope (Leica Microsystems) at 20x magnification in a blinded fashion.

Embryos were embedded in paraffin and sectioned at 7 μm as described in O’Neill et al., 2007 (link). Sectioned embryos were stained with Masson’s Trichrome as per Witten and Hall, 2003 (link) or Mayer’s Hematoxylin and Eosin by clearing in two rinses of Histosol (National Diagnostics), rehydration through serial dilutions of ethanol, staining for 15 min in Mayer’s Haematoxylin (Sigma), rinsing in running tap water for 20 min, rinsing briefly in 95% ethanol, and staining in 0.1% w/v Eosin Y (Sigma) in 95% ethanol for 2 min. Slides were then washed briefly in 95% ethanol, washed briefly with 100% ethanol, cleared with Histosol, and mounted with permount (Sigma).