Fig. 13
Left panel. Representative 10× (upper panel) and 20× (bottom panel) magnification micrographs of the bottom layer of Hematoxylin and Eosin (H&E)-stained histological section of male rat’s middle right-side BEFAF segment 7 days post-surgery, where both flaps were subjected to 4 h PI, 2 h REP1 and 4 h of SI, namely, VO (left flap) or AO (right flap). The deep fascia layer underneath Panniculus carnosus contains the granulation tissue (GT). GT thickness (indicated by yellow arrows) and vascularity (best visible in the 20 × zoom image of yellow rectangular region) will vary depending on the flap harvest time and the severity of the ischemic insult. Scale bar = 200 µm. Right upper panel. Representative 20 × magnification micrograph of the bottom layer of Masson’s Trichrome (MT)-stained histological section of proximal right-side BEFAF segment next to the pedicle 5 days post-surgery where both flaps were subjected to 4 h of PI, 2 h (right flap) or 6 h (left flap) REP1 and 4 h of SI (AVO). Both flaps survived 100% and had functional pedicles. At this magnification, the perfused blood vessels could be examined and counted manually. Bigger collagen-enclosed formations are regions of sprouting angiogenesis and neovasculogenesis. MT stain: blue color = collagen connective tissue fibers; dark red color – muscle and keratin; bright red color – red blood cells (erythrocytes); dark purple/black – cell nuclei; purple – cytoplasm; white – adipose (fat) cells. Bottom panel. The representative 20 × magnification micrograph of PECAM-1 (CD31) positive-vessels. 10% formalin-fixed 4 micron cut paraffin sections of the embedded distal segment of male SD rat flap 7 days after the BEFAF surgery, where the flaps were exposed to either 2 h or 6 h of PI, were stained with recombinant rabbit polyclonal anti-CD31 antibody (RM1006) (Abcam, #ab281583) at 1/400 dilution after heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0. Brown color reaction was detected with chromogen diaminobenzidine (DAB). Scale bar = 200 µm