Rat anti mouse gr 1

For immunofluorescent staining of macrophages, neutrophils, and myofibroblasts, 8μm frozen tissue sections were air-dried, fixed in cold acetone for 10 min, and blocked with 10% goat serum for 30 min. Sections were then incubated with rat anti-mouse CD68 (Abcam, Cambridge, MA), rat anti-mouse Gr-1 (BD Biosciences, San Jose, CA), rabbit anti-mouse α-smooth muscle actin (α-SMA) (for myofibroblast staining, Abcam, Cambridge MA) or rabbit anti-mouse CD11b (Abcam, Cambridge, MA) for 45 min followed by incubation with Alexa fluor 594 goat anti-rat IgG, Alexa fluor goat anti-rat or rabbit IgG 488 (Invitrogen, Carlsbad, CA), respectively. The staining procedures were all performed at room temperature. Stained sections were evaluated using a fluorescence microscope, Axioskop 40 (Zeiss, Oberkochen, Germany) and recorded with a digital camera, AxioCam MRc (Zeiss, Oberkochen, Germany). Gr-1 positive cells in the wounds and wound margins were counted and the average number per 20× field was calculated. The density (% positive staining in wound margin and wound bed) of CD68 and α-SMA was quantified using Image J.^{21 (link)} N = 6 in each group.