Xylene

Slides were deparaffinised in xylene (Electron Microscopy Sciences, Hatfield, PA, USA) and hydrated in 100, 95, and 70% ethanol gradient followed by water.

After the explantation, formalin-fixed grafts were dehydrated in isopropanol during 30 h at 4°C, rinsed in distilled water, embedded in paraffin (Electron Microscopy Sciences), sectioned (5 μm), and finally mounted on glass microscope slides. For a deparaffinization, paraffin-embedded tissue sections were heated in dry oven at 60°C for 20 min, immersed in the following reagents: 3x xylene (Electron Microscopy Sciences) for 10 min, 100, 95, 70, 50, 30% ethanol for 1 min each, physiological saline for 2 min, PBS for 2 min, and finally rinsed with tap water. For hematoxylin and eosin (H&E) staining, the sections were immersed in Harris Hematoxylin solution (Electron Microscopy Sciences) for 1 min, rinsed with tap water, immersed in 1% aqueous Eosin Y solution (Electron Microscopy Sciences) for 1 min, rinsed with tap water, dehydrated in ascending ethanol solutions (50, 70, 80, 2x 95%, and 2x 100%), and then cleared 2x with xylene. Coverslips were mounted onto a labeled glass slide with Permount (Electron Microscopy Sciences). After the staining, sections were evaluated by light microscopy (Axio Imager A1, Carl Zeiss) in a blinded fashion; three sections per stain were assessed from each rat.