2500 alto cryo system

Confocal microscopy was performed on ObaGel- and silk-based in vitro adipogenic differentiated constructs (see Adipogenic Differentiation) and explants following 6-week in vivo implantation and staining with BODIPY lipophilic dye. Confocal research was conducted using a Nikon A1 LASER Ti-E microscope system. The tissue was imaged in Nunc 35 mm glass bottom dishes, at 10x and 20x oil-free objectives, using the FITC and DAPI LASERs. Cryoscanning electron microscopy was conducted on ObaGel constructs and silk scaffolds that were seeded with human SVF cells following a 2-week adipogenic differentiation. Cryo-SEM structure research was done with Gatan 2500 alto Cryo-system and Hitachi S-4800 SEM. The tissue was cut to 7 × 5 × 5 (mm) and laid about 5 mm high above a cryogenic SEM sample holder surface, clamped and glued into the holder, and then frozen in slushed liquid nitrogen at approximately -210°C for about 30 seconds. The frozen tissue was transferred in vacuum to the prechamber attached to the SEM and then fractured and sublimed for 5 minutes at -95°C. After coating for 88 seconds at -130°C with Pt/Pd, the sample was moved to the SEM and observed at 3 kV at -130°C.