Immediately after surgical excision, the fibrovascular tissue was fixed with 2.5% GA, 2% FA, 2mM CaCl2 in 0.1 M Na-cacodylate buffer, pH 7.4 for 2 hours at room temperature. After fixation, the fixative was replaced with fixative including 2% FA in the same buffer prior to preparation of the specimen for SBF-SEM with double osmication. Image series were acquired with FEG-SEM Quanta 250 equipped with a microtome, using a backscattered electron detector. The imaging was done using 0.3-Torr pressure, 2.5 kV beam voltage and spot size 3. Image processing, segmentation and visualization were done using MIB and Amira.

2018