Blocking reagent

Formalin-fixed, paraffin-embedded sections were prepared using femoral artery 7 days after cuff placement. Endogenous peroxidase was blocked by incubation in 3% H₂O₂ for 15 min, and nonspecific protein binding was blocked by incubation for 10 min in Blocking Reagent (Nichirei Bioscience Inc., Tokyo, Japan). The sections were incubated overnight at 4°C with the primary antibody, anti-proliferating cell nuclear antigen (PCNA) antibody (Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK), and phosphorylated extracellular signal-regulated kinase (ERK) and total ERK antibodies (Cell Signaling Technology, Pickering, ON) and Alexa Fluor 488-conjugated F4/80 (BioLegend Inc., San Diego, CA) and R-PE-conjugated LY-6G/-6C (Hycult Biotech Inc., Plymouth Meeting, PA). Antibody binding was visualized by followings: 1) a Zeiss Axioskop2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a computer-based imaging system for 3, 3’-diaminobenzidine (DAB) staining using a detection kit, Histofine (Nichirei Bioscience Inc.) for PCNA and ERK staining, and 2) a fluorescence microscope (Keyence BZ-9000, Osaka, Japan) equipped with a computer-based imaging system for immunofluorescent staining.

Immunofluorescence staining was performed using antibodies against the following antigens: αSMA (1:500; ab32575, abcam, Cambridge, UK) and KLF4 (1:100; JF98-08, Novus Biologicals). Macrophages and IMFs grown on a glass plate were fixed with 4% paraformaldehyde and then incubated with PBS containing 0.2% Triton X-100 and blocked with a blocking reagent (Nichirei Bio Science, Tokyo, Japan). The samples were then reacted with primary antibodies at room temperature for 1 h, washed with PBS, and then incubated with secondary antibodies at room temperature for 1 h. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI). Furthermore, macrophages were attached to glass slides. Confocal images were captured using a FV1000 laser scanning microscope (Olympus Life Science, Tokyo, Japan).