2.5 Tissue Preparation

Excised tissues with hydrogels were fixed in 10% normal buffered formalin (NBF, Fisher Scientific, Waltham, MA) for 24 hr at 4°C then manually dissected into 3–5 mm thick sections, placed in NBF, and fixed for an additional 24 hr at 4°C. Samples were then transferred into Optimal Cutting Temperature (OCT) Compound (Tissue-Tek, Sakura Finetek, Torrance, CA) and left at 4°C for 72 hr. Afterwards tissues were immersed in OCT Compound in plastic molds, then placed in an acetone/dry ice bath for 30 min. Embedded tissues were stored at −80°C until sectioning. For sectioning, embedded molds were equilibrated at their cutting temperature (−22°C) for 30 min then sectioned (10 μm thickness) and mounted on Superfrost Plus glass slides (Electron Microscopy Sciences, Hatfield, PA). Mounted sections were stored at −80°C. Before staining, sections were brought to room temperature and dried overnight.