Tracking Leukocytes in Wound Beds

During the preparation of PRP and PRF, high quantities of platelets and leucocytes are in the buffy coat or embedded in the fibrin clot after centrifugation. To determine whether these leucocytes remained in the wound beds until the end of study, we used DsRed-transgenic mini-pigs as blood donors [40 (link)], and then, the PRP, PRF and PP prepared from them were applied to the full-thickness wounds created on non-transgenic littermate recipients with the same blood type. Blood types were confirmed in advance by mixing donor and recipient blood, and in the absence of agglutination, blood types were concluded to be the same. Except for the source of blood, the other procedures are the same as described in the above methods. Skin biopsies of wound beds were obtained on day 14 post-wounding. After adequate fixation and paraffin embedding, histological slices were cut and subjected to immunofluorescence (IF) [48 (link)] and immunohistochemistry (IHC) [49 (link), 50 (link)] staining using rabbit anti-DsRed primary antibody (ab62341; Abcam, Eugene, CA, USA) and FITC-conjugated and peroxidase-conjugated secondary anti-rabbit antibodies (ab97063; Abcam, Eugene, CA, USA), respectively.