Following stimulation, coronary artery endothelial cells (CAEC) express adhesion molecules detectable via immuno-SERS imaging in single and multiplex formats. (A) Fluorescence images of immunohistochemical staining of ICAM-1, VCAM-1 and P-selectin on CAEC in unstimulated and 10 ng/mL TNF-α-stimulated conditions. Isotype control, ICAM-1, VCAM-1 and P-selectin staining shown in green; nuclei were counterstained using Hoechst 33342 (blue). (B) CAEC were stimulated with 10 ng/mL TNF-α for 24 h, fixed in acetone, and incubated with isotype control, anti-ICAM-1, anti-VCAM-1 or anti-P-selectin BFNP or (C) with all BFNP simultaneously before being subjected to SERS mapping. (D) Representative spectra from anti-ICAM-1 (purple), anti-VCAM-1 (red) and anti-P-selectin (blue) BFNP acquired from the color-matched circles in (C) are shown above their respective reference spectra. Optical images in (B-C) are darkfield images. Scale bars = 20 μm. Results are representative of 3 independent experiments.