Intracellular localization of CCL2 in HBMEC by immunogold silver staining. (A) Unstimulated HBMEC constitutively express CCL2 as shown by the positive cytoplasmic staining in the form of fine, granular black deposits of silver-enhanced gold particles. Nuclei are counterstained with Giemsa. (B)-(D) The intensity of staining is markedly increased following incubation with (B) TNF-α (10 U/ml) for 48 hrs, (C) IL-1β (10 U/ml) for 72 hrs and (D) LPS (5 μg/ml) for 48 hrs. (E) Control cultures incubated with secondary antibody only show no staining. Scale bars = 50 μm.