Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues

Abstract

There is shortage of extensive clinicopathologic studies of cellular senescence because the most reliable senescence biomarker, the detection of Senescence-Associated-beta-galactosidase activity (SA-β-gal), is inapplicable in archival material and requires snap-frozen tissues. We validated the histochemical Sudan-Black-B (SBB) specific stain of lipofuscin, an aggregate of oxidized proteins, lipids and metals, known to accumulate in aged tissues, as an additional reliable approach to detect senescent cells independently of sample preparation. We analyzed cellular systems in which senescence was triggered by replicative exhaustion or stressful stimuli, conditional knock-in mice producing precancerous lesions exhibiting senescence, and human preneoplastic lesions known to contain senescent cells. In the above settings we demonstrated co-localization of lipofuscin and SA-β-gal in senescent cells in vitro and in vivo (cryo-preserved tissue), strongly supporting the candidacy of lipofuscin for a biomarker of cellular senescence. Furthermore, cryo-preserved tissues positive for SA-β-gal were formalin-fixed, paraffin-embedded, and stained with SBB. The corresponding SA-β-gal positive tissue areas stained specifically for lipofuscin by SBB, whereas tissues negative for SA-β-gal were lipofuscin negative, validating the sensitivity and specificity of the SBB staining to visualize senescent cells in archival material. The latter unique property of SBB could be exploited in research on widely available retrospective tissue material.

Senescence-associated inflammatory responses: aging and cancer perspectives

“…To overcome this problem, the in vivo detection of senescent cells is usually based on a combination of markers, most common of which is senescence-associated bgalactosidase staining [56], based on the fact that senescent cells are characterized by increased lysosomal content [57]. Recently, it was reported that staining of lysosomal aggregates by Sudan Black could also serve to identify senescent cells [58]. Another common feature of senescent cells is downregulation of cell cycle markers, such as KI67 or low bromodeoxyuridine (BrdU) incorporation, along with upregulation of markers for growth arrest, such as p53, p21, p16, and unphosphorylated Rb.…”

Geroconversion: irreversible step to cellular senescence

“…In rare cases, (stressinduced and oncogene-induced senescence), geroconversion renders cells "typically senescent," resembling senescence in vitro. 5,[106][107][108][109][110][111] A striking example is oncogene-induced senescent nevi. 112 Gerontologists are focused on these "typically" senescent cells.…”

IL-33 Is Required for Disposal of Unnecessary Cells during Ovarian Atresia through Regulation of Autophagy and Macrophage Migration

“…Two methods were used for detection of ovarian lipofuscin. The first method was Sudan Black B staining following a published method (15). Briefly, non-fixed frozen ovarian sections were observed by fluorescent microscope through three channels and autofluorescent images were recorded.…”