Ep3095

After deparaffinization, tissue sections were incubated in a 0.01 m citrate buffer (pH 6.0) at 121°C for 15 min to retrieve the antigenicity of the relevant proteins. The sections were then immersed in 0.3% H₂O₂ and 0.1% sodium azide (Wako Pure Chemical Industries) in phosphate‐buffered saline (1/15 m, pH 7.4) for 25 min to block endogenous peroxidase activity. After incubation in 10% normal goat serum (Vector Laboratories), sections were treated with antibodies against podoplanin (1:100, D2‐40; Dako) and CD31 (1:250, EP3095; Abcam) at room temperature overnight. After washing in phosphate‐buffered saline, sections were incubated with alkaline‐phosphatase‐conjugated anti‐mouse IgG (Histofine Simple Stain AP, Mouse; Nichirei Bioscience) for 1 h at room temperature as a secondary antibody for podoplanin immunostaining. The blue immunoreaction was visualized using an alkaline phosphatase reaction (Vector blue substrate kit, Vector Laboratories). Sections were then incubated with peroxidase‐conjugated anti‐rabbit IgG (Histofine Simple Stain MAX‐PO, Rabbit; Nichirei Bioscience) for 1 h at room temperature. The brown immunoreaction for CD31 was visualized using 3,3'‐diaminobenzidine reaction (Wako Pure Chemical Industries). The stained sections were examined using a BX‐60 light microscope equipped with a DP72 digital imaging system (Olympus).

The entire RCA and LAD were collected, and the proximal 30 mm fragment of RCA and LAD was further cut onto six 5 mm fragments for embedding in paraffin. Histological blocks were sequentially labeled 1–6; serial 6 μm cross sections were cut from each block and stained with Gomori’s trichrome stain (Polysciences Inc). Sections from each block were used for morphological analysis. EEM, internal elastic membrane, and luminal border were manually outlined in CellSens Dimension 1.18 software (Olympus Corp), and corresponding CSAs were measured by 2 independent researchers, 1 of them under a blinded protocol. The cellular content of coronary plaques was assessed by IHC with serial sections obtained from the middle (no. 3) coronary fragment. Cell marker–specific antibodies for α-SMA (MilliporeSigma, CBL171, clone ASM1), MSR (TransGenia Inc, KT022, clone SRA-E5), and CD31 (Abcam, 134168, clone EP3095) were used for IHC to identify SMCs, MFs, and ECs, respectively. Proliferating cells and cells with DNA damage were quantified by IHC with antibody against PCNA (MilliporeSigma, MAB424R, clone PC10) and pH2A.X (Abcam, ab2893), respectively. Cell apoptosis was quantified with In Situ Cell Death Detection Kit, TMR red (MilliporeSigma, 12156792910), as per manufacturer’s instructions.