Hematoxylin and Eosin Staining
At the 7th POD, six tissue specimens from the flap II region (central
tissue) measuring 1cm × 1cm were collected after the rats were
sacrificed with an excess sodium pentobarbital dose. After 24 hours of
fixation in 4% (v/m) paraformaldehyde, these specimens were embedded in
paraffin wax and transversely sectioned (4 mm thickness). We randomly
selected the number of blood vessel cross-sections per unit area in
different fields of view (/mm2) under the microscope
(Olympus Corp, Japan) to compute the mean blood vessel density of the
flap tissue in order to evaluate the microcirculation level.