at 4 C for at least 24 h followed by transfer to 70 % ethanol
37. The tissue sections can be counterstained with hematoxylin to visualize nucleus
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Acknowledgment
This work was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2058845) to K.H.B. and by R01CA118374, The Garrett B. Smith Foundation and the TedDriven Foundation to S.R.
References
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Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_19, © Springer Science+Business Media New York 2017
Chapter 19
Detection of Senescence Markers During Mammalian Embryonic Development
Mekayla Storer and William M. Keyes
Abstract
Senescence-associated β-galactosidase (SAβ-gal) is a convenient histological technique used to identify senescent cells. Its ease of use is helpful to initially screen and detect senescent cells in heterogeneous cell populations both in vitro and in vivo. However, SAβ-gal staining is not an unequivocal marker of the senescent state, and diagnosis of such usually requires additional markers demonstrating an absence of proliferation and expression of cell-cycle inhibitors. Nonetheless, SAβ-gal remains one of the most widely used biomarkers of senescent cells. Recently, by measuring SAβ-gal activity, the expression of the cyclin- dependent kinase inhibitor p21 (waf1/cip1) and demonstrating a lack of proliferation, we identifi ed senes- cent cells in the developing embryo. This chapter describes the methods for identifying cellular senescence in the embryo, detailing protocols for the detection of SAβ-gal activity in both sections and at the whole mount level, and immunohistochemistry protocols for the detection of additional biomarkers of senescence.
Key words Cellular senescence , SAβ-gal , Whole-mount staining , Embryo , Biomarker , Development , Limb , Neural tube , p21 , Apoptosis
1 Introduction
Cellular senescence is an irreversible form of cell cycle arrest that lim- its the proliferative potential of cells. Initially, senescence was identi- fi ed in primary human fi broblasts that had undergone replicative exhaustion [ 1 ]. Subsequent studies demonstrated that senescence could be induced by a wide variety of stimuli including oncogenic signalling, DNA-damage , oxidative stress, and chemotherapeutic drugs [ 2 – 4 ]. In addition to the permanent arrest of cell division, senescent cells exhibit activation of tumor suppressor networks and an altered pattern of gene expression [ 5 – 9 ]. Historically, however, while cellular senescence has been best characterized as a tumor sup- pressive mechanism and a driver of cellular and tissue aging, recent discoveries have extended its known roles, to include benefi cial effects during embryonic development and tissue repair [ 10 – 20 ].
Twenty years ago, it was discovered that senescent cells express a β-galactosidase activity that is detectable at a suboptimal pH of 6.0 [ 21 ]. This fi nding introduced a quick and easy way of screen- ing for senescent cells in vitro and in vivo, with the enzymatic activity being distinct from the acidic β-galactosidase activity that is present in all cells at pH 4.0. The appropriately named “Senescence- Associated β-galactosidase” (SAβ-gal) was detected using a cyto- chemical assay based on the production of an insoluble blue-dye precipitate that results from the cleavage of X-gal by endogenous β-galactosidase, and which appears to be independent of DNA syn- thesis [ 21 ]. Later, it was elucidated that SAβ-gal activity is due to an increase in the abundance of lysosomal enzymes, possibly linked to an increase in lysosomal biogenesis [ 22 ]. Subsequently, it was shown that the increased activity of lysosomal β- D -galactosidase in senescent cells was a direct consequence of increased levels of GLB1 mRNA and protein [ 23 , 24 ]. Since then, the presence of SAβ-gal activity has been used extensively to identify senescent cells in a variety of conditions, including in cells in culture reaching the end of their lifespan or undergoing premature senescence, or in vivo in response to a variety of senescence-inducing stimuli [ 11 – 13 , 18 , 20 , 25 , 26 ]. However, a note of caution has to be included, as SAβ-gal alone is insuffi cient as a unique identifi er of senescence, as there are incidences where nonsenescent cells stain positive or where SAβ-gal activity is not present in senescent cells [ 21 , 27 , 28 ]. As such, the identifi cation of the senescent state requires the use of additional markers to confi rm the status, includ- ing an absence of markers of cell proliferation such as BrdU or Ki67 , and the expression of hallmark regulatory cell-cycle inhibitors such as p16, p53 , or p21.
Here, we describe general methods to detect SAβ-gal and the senescence marker p21 during embryonic development that can be utilized on both chick and mouse embryos with consistent results.
2 Materials
Prepare all solutions using ultrapure water and store all reagents at room temperature unless otherwise stated.