*4.6. Quantitative RT-PCR*

Total RNA was extracted using TRIzol ® Regent (Invitrogen). cDNA was synthesized using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). qRT-PCR was performed by using a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) with PowerUp SYBR Green master mix (Bio-Rad). GAPDH served as the internal control. The primer pairs used are shown in Table 1. Relative expression was calculated using the 2−ΔΔ*C*<sup>t</sup> method with correction for di fferent amplification e fficiencies.


**Table 1.** The primers used for quantitative real-time RT-PCR.

#### *4.7. Immunohistochemistry, Western Blot Analysis, and Scar Thickness Analysis*

Para ffin-embedded sections were stained with H&E or Masson's trichrome stain and primary antibodies against F4/80, Ki67, and CD34. The immunostained sections were developed with VECTASTAIN Universal Elite ABC Kit (Vector, Burlingame, CA, USA). All antibodies were from Abcam (Cambridge, UK). The sections were analyzed using ImageJ. The excised wound tissue was homogenized with liquid nitrogen. Total protein was isolated using 1% NP-40. Equal amounts of protein were separated by SDS-PAGE and then transferred to a nitrocellulose membrane. The membranes were incubated with primary antibodies against V5 (Invitrogen), VEGF, FGF-2 (Santa Cruz Biotechnology, Dallas, TX, USA), IGF-1 (Abcam), or GAPDH (Cell Signaling Technology, Danvers, MA, USA), followed by horseradish peroxidase-conjugated IgG against mouse, rabbit, or goa<sup>t</sup> antibodies (Jackson ImmunoResearch, West Grove, PA, USA). The membranes were developed by using SuperSignal Chemiluminescent Substrates (Thermo Fisher Scientific, Cambridge, MA, USA). The antibodies used are summarized in Table 2. The quantification was performed by using ImageJ. Scar thickness was measured by photographing the sectioned tissues after Masson's trichrome staining and then using ImageJ.


**Table 2.** The primary antibodies used for this study.
