Immunostaining for DNA modifications: computational analysis of confocal images
For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-met...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
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Journal of Visualized Experiments
2017
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| Online Access: | https://eprints.nottingham.ac.uk/42816/ |
| _version_ | 1848796575107645440 |
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| author | Ramsawhook, Ashley Lewis, Lara C. Eleftheriou, Maria Abakir, Abdulkadir Durczak, Paulina M. Markus, Robert Rajini, Seema Hannan, Nicholas R.F. Coyle, Beth Ruzov, Alexey |
| author_facet | Ramsawhook, Ashley Lewis, Lara C. Eleftheriou, Maria Abakir, Abdulkadir Durczak, Paulina M. Markus, Robert Rajini, Seema Hannan, Nicholas R.F. Coyle, Beth Ruzov, Alexey |
| author_sort | Ramsawhook, Ashley |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans. |
| first_indexed | 2025-11-14T19:50:09Z |
| format | Article |
| id | nottingham-42816 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T19:50:09Z |
| publishDate | 2017 |
| publisher | Journal of Visualized Experiments |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-428162020-05-04T18:54:48Z https://eprints.nottingham.ac.uk/42816/ Immunostaining for DNA modifications: computational analysis of confocal images Ramsawhook, Ashley Lewis, Lara C. Eleftheriou, Maria Abakir, Abdulkadir Durczak, Paulina M. Markus, Robert Rajini, Seema Hannan, Nicholas R.F. Coyle, Beth Ruzov, Alexey For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans. Journal of Visualized Experiments 2017-07-09 Article PeerReviewed Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth and Ruzov, Alexey (2017) Immunostaining for DNA modifications: computational analysis of confocal images. Journal of Visualized Experiments (127). e56318/1-e56318/7. ISSN 1940-087X Epigenetics DNA oxi-mCs N6-methyladenine Immunohistochemistry Zen Confocal Microscopy Signal Quantitation Spatial Distribution https://www.jove.com/video/56318/immunostaining-for-dna-modifications-computational-analysis-confocal doi:10.3791/56318 doi:10.3791/56318 |
| spellingShingle | Epigenetics DNA oxi-mCs N6-methyladenine Immunohistochemistry Zen Confocal Microscopy Signal Quantitation Spatial Distribution Ramsawhook, Ashley Lewis, Lara C. Eleftheriou, Maria Abakir, Abdulkadir Durczak, Paulina M. Markus, Robert Rajini, Seema Hannan, Nicholas R.F. Coyle, Beth Ruzov, Alexey Immunostaining for DNA modifications: computational analysis of confocal images |
| title | Immunostaining for DNA modifications: computational analysis of confocal images |
| title_full | Immunostaining for DNA modifications: computational analysis of confocal images |
| title_fullStr | Immunostaining for DNA modifications: computational analysis of confocal images |
| title_full_unstemmed | Immunostaining for DNA modifications: computational analysis of confocal images |
| title_short | Immunostaining for DNA modifications: computational analysis of confocal images |
| title_sort | immunostaining for dna modifications: computational analysis of confocal images |
| topic | Epigenetics DNA oxi-mCs N6-methyladenine Immunohistochemistry Zen Confocal Microscopy Signal Quantitation Spatial Distribution |
| url | https://eprints.nottingham.ac.uk/42816/ https://eprints.nottingham.ac.uk/42816/ https://eprints.nottingham.ac.uk/42816/ |