Entellan mounting medium

Cells were counted and cytospun onto poly-L-Lysine coated slides (Shandon Cytoslides, Thermo Scientific) at 800 rpm for 3 minutes (Shandon Cytospin 2). Eosinophils were then differentiated by cytological staining using a commercial kit (Diff-Quik, Polysciences, Warrington, PA) per the manufacturer’s instructions. Finally, slides were mounted with Entellan Mounting Medium (Electron Microscopy Sciences).

SRA segments were collected at the end of experiments into optimal cutting compound (ProSciTech), frozen in cold isopentane (Sigma) and stored in −80 °C until required. The renal arteries were used as a landmark to position the tissue and serial sections were taken from the renal arteries to the region of maximal dilatation. Five SRAs were randomly selected from the vehicle, fondaparinux, and dabigatran groups using an online random number generator (https://www.random.org/). Four serial sections of 6 μM thickness were placed on poly-L-lysine coated slides, air dried and subsequently fixed in acetone for 10 min at −20 °C. Adjacent sections were stained with hematoxylin (Polysciences Inc., #24245) and eosin (Polysciences Inc., #09859) (H&E) and mounted in Entellan mounting medium (Electron Microscopy Sciences, #14800) to assess gross morphology. Verhoeff Van Giessen (VVG) staining (Polysciences Inc., #25089) was performed to assess the integrity of SRA elastin. Sections stained with H&E and VVG were photographed using a Nikon Eclipse 50i microscope fitted with a CCD Camera (DSFi1). Digital images were captured to a PC supported with NIS Elements (version F2.30) and analysed as previously described42 (link). Histological evaluations were carried out in a blinded fashion in 3–4 sections per sample (mean coefficient of variation (CV) = 2.97%; n = 5).