Identification of Cells with Markers of Cellular Senescence in Human Formalin-fixed, Paraffin-embedded Brain Tissue Sections

Summary

We describe an immunofluorescence procedure to detect markers of cellular senescence in human formalin-fixed, paraffin-embedded brain sections. In contrast to immunohistochemistry, immunofluorescence permits semi-quantitative measurements and multiple staining on the same slide, which allows detecting different cell types more rigorously and assessing the abundance of senescence markers more objectively.

Abstract

Cellular senescence is a terminal arrest in proliferation associated with aging and age-related diseases. Senescent cells are characterized in vitro by several morphological and biochemical markers, such as flattened morphology, Senescence-Associated beta-galactosidase activity, chromatin rearrangements, and expression of the cyclin-dependent kinase-inhibitors p21 and p16^INK4A.

Here we describe a procedure to detect p16^INK4A-positive cells by immunofluorescence on formalin-fixed, paraffin-embedded, human-brain specimens. Briefly, tissue slides are deparaffinated, rehydrated, and subjected to antigen-retrieval with standard immunohistochemistry procedures. The slides are then incubated with serum to block non-specific binding sites, and with primary antibodies against p16^INK4A and a specific cell type marker. Finally, the slides are stained with fluorescently-conjugated secondary antibodies and counterstained with DAPI. This technique has been used successfully to detect p16^INK4A positive astrocytes in human brains, but can be easily applied to other cell types, markers, and tissues, provided no cross-reactivity ensues between the antibodies.

Our technique allows for the identification of subpopulations of senescent cells more rigorously than immunohistochemistry and enables the user to measure the abundance of a senescence marker semi-quantitatively.

Introduction

Cellular senescence is a terminal arrest in cell proliferation ¹ in response to telomere attrition, DNA damage, oncogenic activation, and oxidative stress ^2,3 . Cells expressing markers of senescence have been found in vivo in skeletal muscle, lung and liver of old animals ⁴ and recently we have demonstrated the presence of senescent astrocytes in human brain ⁵. Senescent cells contribute to the aging process and to the onset of age-related pathologies by secreting several pro-inflammatory cytokines ⁶, a phenomenon known as Senescence-Associated Secretory Phenotype (SASP) ⁷. At the cel....

Protocol

Human Subjects Ethics Statement

This research was performed in compliance with and following approval by the Institutional Review Board at Drexel University College of Medicine (Protocol Number: 18172), and the Institutional Review Board at University of Pennsylvania (Protocol Number: 180600). All tissue samples were de-identified and no protected health information was made available to the researchers; therefore, this protocol was approved as exempt study and consent was waived.

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Discussion

Double immunofluorescence labeling of human brain tissues is a well-established method in the study of neurodegenerative disease ^13-15 and methods for performing immunofluorescence on FFPE tissues have been described previously ¹⁶, thus we redirect the reader to them for a more detailed discussion of the technique, while we will focus on key concepts necessary for applying the technique successfully to detect senescent cells. Background signal and antibody specificity are the most critical ch.......

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
Xylene	Fisher Scientific	X3P-1GAL	histological grade, flammable
Reagent Alcohol	Fisher Scientific	A962F-1GAL	flammable liquid
Citric acid monohydrate	Sigma Aldrich	C7129-500G	eye irritant
Phosphate-buffered Saline	Mediatech	46-013-CM	10X
Bovine Serum Albumin	Gemini Bioproducts	700-101P
Triton X-100	Sigma Aldrich	T-9284	irritant
ImmEdge Pen (PAP pen)	Vector Laboratories	H-4000
Goat Serum	MP Biomedicals	191356
Donkey Serum	Millipore	S30-100ML
Mouse anti p16 antibody	Santa Cruz Biotechnology	sc-1661
Rabbit anti GFAP antibody	Millipore	AB5804
Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)	Life Technologies	A-11008
Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L)	Life Technologies	A31570
DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride)	Sigma Aldrich	32670-5MG-F
Vectashield Mounting Medium	Vector Laboratories	H-1000
Coverslips	Fisher Scientific	12-544-14	24x50 mm
Clear nail polish	Any supplier
Heat steamer	Any supplier		rice cooker
Olympus BX61 fluorescence microscope	Olympus
Hamamatsu ORCA-ER camera
Slide Book 4 Software	Intelligent Innovations, Inc	version 4.0.1.44