Anti neutrophil antibody

Mice were euthanized via the IP injection of a lethal dose of pentobarbital sodium on days 3, 7, 11, or 14 after wounding. The wound and the surrounding intact skin were harvested, and each sample of wound tissue and the surrounding intact skin was bisected at the center of the wound. The wound samples were stapled onto polypropylene sheets to prevent overcontraction, before being fixed in 4% paraformaldehyde for 18 hours. The samples were then dehydrated in a graded alcohol series, cleaned in xylene, and embedded in paraffin, before 5-μm serial paraffin sections were prepared. At least 6 serial sections from near to the center of the wound were obtained per wound and stained according to the following methods [17 (link)]. Five-μm thick sections were subjected to hematoxylin-eosin (HE) or azan staining or were immunohistologically stained with anti-neutrophil antibody to detect neutrophils (1 : 100; Abcam Japan, Tokyo, Japan), anti-Mac-3 antibody to detect macrophages (1 : 100; BD Pharmingen, Tokyo, Japan), or anti-α-smooth muscle actin (α-SMA) antibody to detect myofibroblasts (1 : 300; Abcam Japan, Tokyo, Japan). Negative control slides were obtained by omitting each primary antibody.

YC-1, ɑ-SMA and β-actin antibodies were from Sigma-Aldrich (St. Louis, MO); TNF-ɑ, and IL-6 kits were from R&D Systems (Minneapolis, MN); serum alanine aminotransferase kit and ECL were obtained from Amersham (Piscataway, NJ); anti-neutrophil antibody was from Abcam (Cambridge, MA); vWF antibody was from Thermo Scientific (Rockford, IL), F4/80 was from Biolegend; anti-HIF-1ɑ antibody was from Santa Cruz; VCAM-1 antibody was from Millipore (Billerica, MA); DAPI antibody was obtained from Invitrogen (Carlsbad, CA); anti-VEGFR1 antibody was from Epitomics (Burlingame, CA).