1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, The University of Melbourne, 4Department of Allergy and Immunology, Murdoch Children's Research Institute, Royal Children's Hospital, 5Department of Pediatrics, The University of Melbourne
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Tang, M. M., Mah, L., Vasireddy, R. S., Georgiadis, G. T., El-Osta, A., Royce, S. G., et al. Quantitation of γH2AX Foci in Tissue Samples. J. Vis. Exp. (40), e2063, doi:10.3791/2063 (2010).
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's Research Institute which adheres to the Australian Code of Practice for the care and use of laboratory animals for scientific purposes.
At this point slides may be stored in sealed containers at 4°C for up to 2 weeks.
Microscopy / Analysis
Images were acquired in a Z-series pattern with a step size of 0.5 μm. During analysis, individual planes were deconvoluted and stacked to produce a maximum projected image to minimize the overlap of foci.
The method of creating a maximum projected image prior to a particle count should only be used in circumstances where a thin section (e.g. 5μm) is used and where the background staining is insignificant. Where thicker sections with a significant background must be analyzed, a more complex analysis such as 3D Object Counter as described by Bolte and Cordelieres (2006) should be used7.
Although Metamorph can be used to quantitate foci numbers, typically when using tissue sections foci are counted manually (by eye) for greater accuracy.
Specific cell types of interest can be selected for quantitation, for instance lung epithelial cells. This may be done based on histology and with reference to haematoxylin and eosin stained serial sections.
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 DSB-inducing agent, for example naphthalene, which is known to induce DSBs in the mouse lung model, would yield higher foci numbers. Indeed, this protocol is most suited for the investigation of the effects of ionizing radiation in tissues; more striking results, in terms of γH2AX foci numbers and resolution are obtained when ionizing radiation is used. It is well known that following exposure to X-rays, γH2AX foci form rapidly and foci numbers reach a maximum between 30-60 minutes2. Therefore, 1 hour time points (post-irradiation) are ideal for evaluating initial DSB formation and longer times (typically up to 48 hours) can be used to monitor DNA repair.
Overall, quantitation of γH2AX in tissues is useful for monitoring DSB formation and repair. Apart from its utility in the context of ionizing radiation, the method can be applied to evaluation of the efficacy of DSB-inducing compounds in various tissues, for example the commonly used anticancer anthracyclines such as doxorubicin are known to cause DSBs, and potentially to monitor various pathologies that are characterized by reactive oxygen species-mediated DSBs. Importantly, this protocol is suitable for different mouse tissues.
No conflicts of interest declared.
The support of the Australian Institute of Nuclear Science and Engineering is acknowledged. TCK was the recipient of AINSE awards. Epigenomic Medicine Lab is supported by the National Health and Medical Research Council of Australia (566559). LM is supported by Melbourne Research (University of Melbourne) and Biomedical Imaging CRC supplementary scholarships. The support of Monash Micro Imaging (Drs Stephen Cody and Iśka Carmichael) was invaluable for this work. MMT is grateful for the expert assistance and advice from Dr Olga Sedelnikova from the National Institutes of Health.
|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.|
|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|
|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|
|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|