Research Paper Volume 5, Issue 1 pp 37—50
Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues
Figure 3. Lipofuscin accumulates and co-localizes with Senescence-Associated beta-galactosidase (SA-β-gal) in p53-mediated Saos-2 senescent cells. (A) Senescent cells with the characteristic morphology (enlarged and flattened) and positivity for SA-β-gal staining (turquoise color), on the 8th day of induced with doxycycline of the Saos-2 p53 Tet-On system. (B) Sudan Black B (SBB) perinuclear accumulation as dark blue-black granules, in cells with senescent morphology. (C) Top panels: Perinuclear appearance of lipofuscin in apparently senescent cells: pseudocolor visualization of lipofuscin's auto-fluorescence (450-490 nm) is represented in green. Bottom panels: Lipofuscin's auto-fluorescence (FM, fluorescence microscopy) is masked by SBB staining (BF, bright field microscopy). (D) Co-localization of SA-β-gal and SBB staining in senescent cells and, (E) Ki67 positive cells are negative for SBB and SA-β-gal. (F) Addition of 5μg/ml doxycyclin (Dox) leads to p53 expression. Brown dashed lines: Ki67- negative nuclei, black arrows: SBB granules, NFR: nuclear fast red counterstain.