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. 2021 Oct 4;35(12):917–950. doi: 10.1089/ars.2020.8234

FIG. 6.

FIG. 6.

Ferryl hemoglobin-positive macrophages in carotid artery are positive for IL-1β and TNF-α. Cross sections of a healthy carotid artery and atheromatous and hemorrhagic transformed lesions were shown. (A) Images demonstrated macrophages positive for both CD68 and IL-1β in the atheromatous plaque and positive for ferrylHb, CD68, and IL-1β in the hemorrhagic transformed lesions. Sections were stained with Hoechst 33258 for DNA (blue), an anti-ferrylHb antibody with Alexa Flour 488 secondary antibody for ferrylHb (green), an anti-CD68 antibody with Alexa Flour 568 secondary antibody for CD68 (yellow), and an anti-IL-1β antibody with Alexa Flour 647 secondary antibody for IL-1β (red). (B) Images demonstrated macrophages positive for both CD68 and TNF-α in the atheromatous plaque and positive for ferrylHb, CD68, and TNF-α in the hemorrhagic transformed lesions. Sections were stained with Hoechst 33258 for DNA (blue), an anti-ferrylHb antibody with Alexa Flour 488 secondary antibody for ferrylHb (green), an anti-CD68 antibody with Alexa Flour 568 secondary antibody for CD68 (yellow), and an anti-TNF-α antibody with Alexa Flour 647 secondary antibody for TNF-α (red). Images were taken using Leica TCS SP8 gated STED-CW nanoscopy. Images were deconvolved using Huygens Professional software. Representative image, N = 5. (C) Fluorescence intensity for ferrylHb (n = 17), CD68 (n = 20), IL-1β (n = 19), and TNF-α (n = 15) staining was calculated using ImageJ software. *p < 0.05; **p < 0.01; ***p < 0.001. Color images are available online.