Supplementary MaterialsS1 ARRIVE Checklist: The ARRIVE Recommendations Checklist. thickness skin samples were collected from the entire wound sites (including the scab and epithelial margins) for evaluation. Full thickness skin samples from sham-operated (non-wounded) mice served as pre-wound controls. The skin samples were divided in two halves- one half fixed and stored in 10% formalin for histological analysis and other half frozen in liquid nitrogen and stored at -80C for qPCR analysis. Measurement of wound closure rate The residual wound area was traced on a transparent film daily after skin excision until day 14 and the pixel of the traced area was analyzed by ImageJ (Software 1.48q, Rayne Rasband, National Institutes of Health, USA). Wound area analysis was done by a different person who was blinded to the groups. Wound area percentage was calculated according to the following formula: Wound area (%) = [(Areaday n) /Areaday 0] 100; where Areaday 0 is the initial wound area at day 0 and the Areaday n is the area on day n after wounding. Histological and immunohistochemistry evaluation The wounded skin tissues were harvested and fixed in 10% formalin and later embedded in paraffin. The tissue blocks were then cut into 5 m sections, transferred to glass slides, and stained with H&E or Masson-Trichrome. Morphologic alterations in the skin tissues were examined by light microscopy and documented by photographs. For immunostaining, paraffin-embedded sections were deparaffinized in xylene and rehydrated in a HA-1077 inhibitor database graded series of ethanol. Antigen retrieval was performed in the citrate-based antigen unmasking solution, pH 6 (Vector Laboratories, Burlingame, CA) at 95C for 15 min. Endogenous peroxidase activity was quenched by exposing HA-1077 inhibitor database to 2% hydrogen peroxide in 60% methanol for 20 min. After blocking with 2% normal goat serum in Tris-buffered saline, the sections were incubated overnight with anti-mouse Gr-1 antibody (BioLegend, San Diego, CA) or anti-mouse MMP-9 antibody (Calbiochem, Gibbstown, NJ), or anti-mouse CD31 antibody (Santa Cruz Biotechnology, Rabbit Polyclonal to Cytochrome P450 2U1 Dallas, TX), followed by biotinylated species-specific secondary antibody (Vector Laboratories, Burlingame, CA). The detection was carried out with VECTASTAIN Elite ABC reagent and DAB (3, 3-diaminobenzidine) HRP (horseradish peroxidase) substrate kit (Vector Laboratories, Burlingame, CA) as per the manufacturers instructions and counterstained with hematoxylin. The immunostaining was examined under a Nikon Eclipse E600 microscope by at least two investigators blinded to the genotype. The HA-1077 inhibitor database number of Gr-1 positive neutrophils and CD31 positive blood vessels with a visible lumen were manually counted in a microscopic field centered on the wound site in the immunostained sections. Neutrophil infiltration and microvascular density were evaluated based on the average number of neutrophils or blood vessels per counting field from 3 wounds per group for each time point. qPCR analysis Total RNA was extracted from skin tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse-transcribed into cDNA using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA, USA). A PCR reaction was carried out in a 24 l final volume containing 0.08 M of each forward and reverse primer, cDNA, and 12 l SYBR Green PCR Master Mix (Life Technologies, Grand Island, NY). Amplification was conducted in an Applied Biosystems 7300 real-time PCR machine (Applied Biosystems) under the thermal profile of 50C for 2 min and 95C for 10 min, followed by 40 cycles of 95C for 15 s and 60C for 1 min. The data was analyzed by the 2-Ct method for relative quantization normalized to mouse -actin.