Quantitation of γH2AX Foci in Tissue Samples

Summary

Quantitation of DNA double-strand breaks on the basis of γH2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of γH2AX foci in tissue samples.

Abstract

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks¹. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)². Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy². Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer³. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo^4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.

Protocol

Tissues

1. Lung tissue, embedded in moulds in optimal cutting temperature (OCT) compound 4583, was sectioned (5 μm) using a CM Leica 1950 cryostat and mounted onto poly-L-lysine slides.

The frozen samples were obtained from Dr Simon Royce at the Murdoch Children's Research Institute. Lung tissue was obtained from untreated Balb/c mice and from a mouse model of chronic allergic airways disease which shows many of the pathological features of human asthma⁶. Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Animal Ethics Committee of the Murdoch Children....

Discussion

For the purpose of this demonstration we used mouse lung tissue from untreated Balb/c mice and from a mouse model of chronic allergic airways disease. We quantitated differences in the number of foci per cell with slightly higher averages observed in the damaged lung compared to untreated sections. Specifically, quantitation indicated 6.5 foci per/cell and 9.4 foci per/cell (manual counting of 20 cells per section), in the untreated tissue and chronic allergic airways disease tissue, respectively. However, the use of a D.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin (BSA)		Sigma-Aldrich	A7906	BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA.
PBS (without Ca2+ and Mg2+)		Invitrogen	17-517Q
Optimal Cutting Temperature (OCT) Compound 4583		Tissue-Tek	4583
Superfrost Plus	Polysine slides	Menzel-Glaser	SF41296SP	Electrostatically attracts frozen tissue sections and reduces the need for additional coatings and adhesives.
Tween 20	Reagent	Calbiochem	655205	Tween 20 (0.5%), is a detergent to permeabilise cells
Triton X-100	Reagent	Sigma-Aldrich	T8787	Triton X-100 (0.1%) used to permeabilise cells.
Paraformaldehyde	Reagent	Sigma-Aldrich	158127
Anti-phospho-γH2AX (rabbit polyclonal antibody)	Primary Antibody	Upstate, Millipore	07-164	Dilution of primary antibody (1:500), in 1% BSA.
Alexa Fluor 488 goat anti-rabbit IgG (H+L)	Secondary Antibody	Invitrogen	A-11008	Dilution of secondary antibody (1:500), in 1% BSA.
Ethanol (70%)		Thermo Fisher Scientific, Inc.	BSPEL316
RNAse A	Reagent	Qiagen	19101
Vectashield PI	Reagent	Vector Laboratories	H-1300	A glycerol based mounting medium containing propidium iodide (a red nuclear stain).This medium must be used with an oil based lense that matches its refractive index. Note: DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser.
Mini PAP Pen		Invitrogen	008877
Coverslips (22x50mm)		Menzel-Glaser	CS2250100
Coplin Jar, glass		Grale Scientific P/L	1771-OG
Staining Trough		Grale Scientific P/L	V1991.99
Zeiss LSM 510 Meta Confocal		Confocal Microscope
CM Leica 1950 Cryostat		Leica Microsystems
Metamorph	Software for Imaging analysis	Molecular Devices