Dual-Color Immunostaining for Atherosclerotic Plaques

The methods used for dual-color immunostaining have previously been described^{29 (link)}. Cells were fixed with 4% paraformaldehyde solution (Affymetrix) in glass bottom tissue culture plates, incubated with primary antibody at 4 °C overnight, followed by fluorescence conjugated secondary antibody at room temperature for 2 hr. For tissue staining, atherosclerotic plaques were cut into 4μm thick sections and permeabilized with 0.5% Triton-X-100 in PBS for 20 min. Tissue sections were incubated with primary antibodies for overnight at 4 °C and secondary antibodies for 1 hour at 37°C. Nuclei were labeled with DAPI (Santa Cruz Biotechnology) for 10 mins at room temperature. Images were analyzed using the Olympus fluorescence microscopy system (Olympus, Tokyo, Japan) or the All-in-One Fluorescence Microscope BZX800E system (Keyence, Kyoto, Japan). Following antibodies were used: CD155 Monoclonal Antibody (Thermo Fisher Scientific, MA5–13493,1:200), CD68 Monoclonal Antibody (Thermo Fisher Scientific,MA5–13324,1:200), METTL3 (E3F2A) Rabbit mAb (Cell Signaling Technology 86132S,1:200), Goat anti-rabbit IgG (H+L), Alexa Fluor 488 (Thermo Fisher Scientific, A-11008,1:200) and Goat anti-mouse IgG (H+L), Alexa Fluor 594 (Thermo Fisher Scientific, A-31635,1:200). Detailed information of all antibodies used are listed in Supplementary Table 2.