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.