*4.6. Histology and Immunohistochemistry*

The tissues were fixed with 4% paraformaldehyde-phosphate buffer solution and embedded in paraffin. Sections were taken from the central portion of the wound and stained with hematoxylin-eosin (HE) according to the standard method.

For immunohistochemical analysis, after endogenous peroxidase was blocked with methanol/hydrogen peroxide, the sections were incubated with 10% normal rabbit serum for 20 min to block non-specific binding and then stained with anti-α-smooth muscle actin (α-SMA) antibody (dilution 1:200; Vector Laboratories, Inc., Burlingame, CA, USA), anti-Ly6G Ab (clone 1A8; dilution 1:100; BioLegend), or anti-MMP-2 (dilution 1:200; Chemicon, Darmstadt, Germany). The sections were incubated with peroxidase-conjugated secondary Ab (4 μg/mL; Histofine Simple Stain MAX-PO, Nichirei Bioscience, Tokyo, Japan), then reacted with 3, 3-diaminobenzidine (DAB) (Nichirei Bioscience) or Alkaline Phosphatase (Dako, Bettingen, Switzerland). The number of myofibroblasts and neutrophils in six random fields (each 0.2 mm2) was determined by counting the number of α-SMA-positive cells or the number of Ly6G-positive cells, respectively. All analyses were performed under blinded conditions.

#### *4.7. RNA Extraction and Quantitative Real-Time RT-PCR*

Total RNA was extracted from the wound tissues using ISOGEN (Nippon Gene Co. Ltd., Tokyo, Japan), and first-strand cDNA was synthesized using the PrimeScript first-strand cDNA synthesis kit (TaKaRa Bio Inc., Otsu, Japan) according to the manufacturer's instructions. Quantitative real-time PCR was performed in a volume of 20 μL using gene-specific primers and FastStart essential DNA green master mix (Roche Applied Science, Penzburg, Germany) in a Step OneTM (Thermo Fisher Scientific, Waltham, MA, USA). Primers were as follows: 5- TGT TCA GCT TTG ACC TCC G -3 (Forward) and 5- TAC CTC GGG TTT CCA CGT CTC A -3 (Reverse) for COL1A1, 5- GGA CCA GGC AAT GAT GGA AAA CC -3 (Forward) and 5- ACC AGG GAA ACC CAT GAC ACC -3 (Reverse) for COL3A1, 5-CCG CGC CTA TCG CCA ATG AGC TGC GC-3 (Forward) and 5-CTT GGG GAC ACC TTT TAG CAT CTT TTG G-3 (Reverse) for CXCL1 (KC), 5-CTG AAC AAA GGC AAG GCT AAC TG -3 (Forward) and 5-CAC ATC AGG TAC GAT CCA GGC TT -3 (Reverse) for CXCL2 (MIP-2), 5- CCC CTG ATG TCC AGC AAG TAG A -3 (Forward) and 5- AGT CTG CGA TGA GCT TAG GGA AA-3 (Reverse) for MMP-2, 5- CCC TGG AAC TCA CAC GAC ATC TTC-3 (Forward) and

5- GGT CCA CCT TGT TCA CCT CAT TTT -3 (Reverse) for MMP-9 and 5-GCT TCC TCC TCA GAC CGC TT-3 (Forward) and 5-TCG CTA ATC ACG ACG CTG GG-3 (Reverse) for β-actin (ACTB). The reaction e fficiency with each primer set was determined using standard amplifications. Target gene expression levels and that of ACTB as a reference gene were calculated for each sample using the reaction e fficiency. The results were analyzed using a relative quantification procedure and are presented as expression levels relative to that of ACTB.
