Fixation and Tissue Tracking
Tissues were fixed with formaldehyde for 48 hours. The tissues were washed with Phosphate-Buffered Saline (PBS) at pH 7.2 / 7.4 after fixation. Tissues were first passed through a series of ethanol increasing from 80% to 95% and 2 times 96% respectively. It was held for 45 minutes in each ethanol series. In the transparentization step, the tissues were xylolized twice for 1 hour. The tissues whose transparency is completed are embedded in paraffin for 2 times for 1 hour and then blocked (14). The tissues were kept at + 4ºC after the blocking process. 5 μm sections were taken with a rotary microtome.