F0250

Histologic sections were processed in the same manner as for enzyme IHC up to the primary antibody reaction. The sections were incubated with alkaline phosphatase‐conjugated secondary antibody followed by the VECTOR Blue Alkaline Phosphatase Substrate Kit III (SK‐5300, Vector Laboratories) or with biotinylated horse secondary antibody (Vectastain Universal Elite ABC Kit), and then incubated with fluorescein isothiocyanate‐conjugated streptavidin (F0250, DAKO). The sections were then microwaved in 10 mmol/L citrate buffer (pH 6.0) for 20 minutes to inactivate the antibodies used for detecting the first antigen, and then incubated with normal horse serum for 10 minutes. Subsequently, the sections were incubated overnight with the appropriately diluted first antibodies against the second antigen at room temperature. The sections were next incubated with EnVision + System‐HRP Labelled Polymer (K4001, DAKO) followed by the Histofine Simplestain DAB Solution, or with tetramethylrhodamine isothiocyanate‐conjugated anti‐mouse immunoglobulin (R0270, DAKO). Double fluorescence IHC was performed using a fluorescence laser‐scanning microscope (FV1200; Olympus).

Immunohistochemical and immunocytochemical analyses were performed to confirm the expression of RAGE, S100A8 and S100A9 in sections of lungs and in PASMCs. For immunohistochemical analysis, lungs from patients with PAH or control subjects were fixed with 4% paraformaldehyde. Immunofluorescence was performed on 5-μm lung slices. For immunocytochemical analysis, PASMCs were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Rabbit anti-human RAGE antibody (Santa Cruz Biotechnology), mouse anti-αSMA antibody (Sigma-Aldrich), goat anti-S100A8 (calgranulin A) antibody (Santa Cruz Biotechnology) and goat anti-S100A9 (calgranulin B) antibody (Santa Cruz Biotechnology) were used. The second antibodies were swine anti-rabbit (FITC, F0205) (Dako), rabbit anti-mouse (TRITC, R0270) (Dako) and rabbit, anti-goat (FITC, F0250) (Dako).