2.7 Protein extraction and western blot analysis
Dorsal skin samples were dissected and stored at -80 °C. Protein lysates were prepared using lysis buffer containing a protease inhibitor, calcineurin inhibitors and PMSF. The concentration of protein in the lysates was measured using the BCA Protein Assay Kit (Beyotime, Shanghai, China) and equal amounts of protein were applied on 10% SDS-PAGE gels. Separated proteins were electrophoretically transferred to PVDF membranes. After being blocked with 5% skim milk, the membranes were incubated with anti-F4/80, CD206, iNOS (Abcam, Cambridge, UK; 1:1000) at 4 ℃ overnight. After washing, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Immunoreactive signals were detected using an enhanced chemiluminescence solution (ThermoScientific, MA, USA).
2.8 Fluorescent immunohistochemistry analysis
Immunofluorescent staining was performed using 4-um paraffin sections. Primary antibodies of F4/80; CD206; iNOS (Abcam, Cambridge, MA; 1:100), and corresponding secondary antibodies (Invitrogen, Carlsbad, CA, USA) were used for staining. Nuclei were stained with DAPI. The images were obtained by a laser scanning microscope system (Nikon Eclipse Ni, Tokyo, Japan).