Fig. 2.

Histological characterization of healing wound tissue used for gene expression profiling studies. Wound (Fig. 1) tissues from excisional wounds were collected at indicated time postinjury. Formalin-fixed paraffin sections or OCT-embedded frozen sections were stained using Masson Trichrome procedure (A). This procedure results in blue-black nuclei, blue collagen and cytoplasm. Epidermal cells are stained in red. i: Low magnification (×1.25) images showing cross-section of entire wounds at 6 and 96 h postwounding. Boxed area shows the wound edge tissue shown in ii and iii. Wound-edge tissue imaged with ×5 (ii) or ×20 (iii) objectives. Scale bar = 200 μm (for ii) and 50 μm (for iii). D, dermis; EGT, early granulation tissue; Es, Eschar tissue; FP, fibrin plug; HE, hyperproliferative epithelium. The newly forming epithelial tip is shown with an arrow. Alternatively, sections were immunostained as shown in B. B, i: Antineutrophil antibody (brown, shown with black arrow) that recognizes a polymorphic 40 kDa antigen expressed by polymorphonuclear cells. Counterstaining was performed using hematoxylin (blue); ii: F4/80 antibody (brown, shown with black arrow) that recognizes the murine F4/80 antigen, a 160 kDa cell surface glycoprotein expressed on a wide range of mature tissue macrophages. Counterstaining was performed using hematoxylin (blue); iii: Cd31 antibody (red fluorescence, shown with white arrow) that recognizes the mouse CD31, a 140 kDa cell surface glycoprotein that is expressed at high levels on endothelial cells. Counterstaining was performed using DAPI (blue, fluorescence). Scale bar, 50 μm. iv: Relative quantification (arbitrary units) of neutrophils, macrophages, and CD31+ endothelial cells from tissue sections obtained 6–96 h postwound was performed using a image processing tool kit. Data are means ± SD (n = 3). *P < 0.05 compared with 6 h time point postwounding.