The work was supported by Biotechnology and Biological Sciences Research Council [BB/N005759/1 to A.R.]. NRFH was funded by The Rosetrees Trust and The Stoneygate Trust [A1130 / M546].&#160;The Zeiss Elyra PS.1 microscope, the processing computers and software were funded by BBSRC BB/L013827/1 (Multidisciplinary Super Resolution Microscopy Facility at Nottingham University grant)

1. Generation of Confocal Images
<ol>
	<li>In preparation for the analysis of modified forms of cytosine, perform immunohistochemical staining as described by Abakir&#160;et al.34.</li>
	<li>Carry out imaging of slides out using a microscope and save files in an LSM format. 
	NOTE: When comparing intensity profiles between samples the values used for laser power and gain for each channel must be maintained throughout the image taking procedure to allow for direct comparison at the image analysis stage.</li>
</ol>
2. Generation of 2.5D Intensity Plots
<ol>
	<li>Open the software (e.g., Zen Black) and select &#39;Image Processing&#39;.</li>
	<li>Go to the File menu and select Open. Select the image that requires processing.</li>
	<li>Click on &#39;Show All&#39; below the image to make all graphical controls visible. In the lower half of the screen there are three tabs; &#39;Dimensions&#39;, &#39;Display&#39; and &#39;Graphics&#39;, select the &#39;Graphics&#39; tab.
	<ol>
		<li>Choose the &#39;rectangle&#39; tool to select nuclei/area of interest.</li>
		<li>Select &#39;Cut region&#39; to crop the image. This will generate a new image for the cropped region in a separate tab.</li>
		<li>Go to the File menu and select Save As. Give the file a name and save it as an LSM or CZI file.</li>
	</ol>
	</li>
	<li>Open software (e.g., Zen Blue) and select the &#39;Zen Desk&#39;.</li>
	<li>Send the cropped image from Zen Black to Zen Blue by selecting the file and clicking on the File menu and select Send to Zen 2012-blue edition. The cropped image will then open in the relevant program. 
	NOTE: The image file is transferred because the 2.5D intensity plot feature in Zen Black 2012 does not display multiple channel images simultaneously.</li>
	<li>On the left hand side of the image choose the tab labelled &#39;2.5D&#39;, this enables visualization of the image in 2.5D. Click &#39;Show All&#39; to make all graphical controls visible.</li>
	<li>Alter visualization settings using four grey bars situated to the left and below the image. 
	NOTE: Left hand bar, top of the screen = zoom. Left hand bar, bottom of the screen = turn image on axis. Bottom of screen, left hand bar = rotation of image. Bottom of screen, right hand bar = expand and contract scale bars.</li>
	<li>Ensure that render mode selected is surface as this gives the best visualization of the individual peaks.</li>
	<li>Alter grid distance by changing the percentage, the lower the percentage the more defined each individual peak.</li>
	<li>To save the 2.5D image, first hide the file list on the right side and the graphical tools below the image by clicking on the grey arrow below the 2.5D plot (next to &#39;show all&#39;), this will hide the graphical tabs allowing for the image to expand to fill the screen. To save the 2.5D plot, press the &#39;PrtSc&#39; key on the keyboard. This will capture a screenshot of the display. Paste the screenshot in Microsoft Paint and crop the image before saving.</li>
</ol>
3. Intensity Profiling
<ol>
	<li>Open the software (e.g., Zen Black) and select Image Processing.</li>
	<li>Go to the File menu and select Open. Select the image that requires processing.</li>
	<li>AS the image will appear on the screen, on the left hand side of the image select the tab labelled &#39;Profile&#39;.</li>
	<li>In the lower half of the screen select (tick) the &#39;Show All&#39; tab. This will enable access to all formatting options.</li>
	<li>In the lower half of the screen there are three tabs; &#39;Dimensions&#39;, &#39;Display&#39; and &#39;Profile&#39;; select &#39;Profile&#39; and select (tick) the &#39;Table&#39; box. Select the &#39;Arrow&#39; button.</li>
	<li>On the image use the mouse to select a start point and drag the mouse over a continuous number of cells. Release the mouse to produce an intensity plot for the cells along the line; additionally the table will be populated by intensity measurement data for the pixels along the line for each wavelength. 
	NOTE: The table will provide the distance (&#181;m) at which pixel intensity is read for the red, green and blue fluorescence.</li>
	<li>To export this data, right click on the table, select &#39;Save table&#8230;&#39; and save to an appropriate location as a .txt file.</li>
	<li>To save the selected intensity profile image select the File menu and select &#39;Export&#8230;&#39;. In the pop-up window choose &#39;Tagged Image File (Format)&#39; and &#39;Contents of Image Window - single pane (Data)&#39; followed by clicking &#39;Select file name and save&#8230;&#39; choose an appropriate location and click &#39;Save&#39;.</li>
	<li>Repeat the process for each individual Intensity profile depending on the fields of vision and number of profiles to analyze.</li>
	<li>Import intensity profiles data saved (see 3.7) and analyze in a spreadsheet followed by statistical analysis.</li>
</ol>
4. Co-localization Analysis
<ol>
	<li>Open the software and select &#39;Image processing&#39;.</li>
	<li>Go to the File menu and select Open. Select the image that requires processing.</li>
	<li>As the image will appear on the screen, on the left hand side of the image select the tab labelled &#39;Coloc&#39;.</li>
	<li>In the lower half of the screen select (tick) the &#39;Show All&#39; tab. This will enable access to all formatting options.</li>
	<li>In the lower half of the screen there are four tabs; &#39;Dimensions&#39;, &#39;Display&#39;, &#39;Graphics&#39; and &#39;Colocalization&#39;; select &#39;Colocalization&#39; and tick the box for &#39;Table&#39; and &#39;Image&#39;.
	<ol>
		<li>Select the third icon along at the top of this tab (Close Bezier text will appear when the mouse hovers over the tool) then use this tool to encircle a single nuclei, values for this nuclei will then appear in the table. 
		NOTE: For each encircled nuclei a scatter plot is produced for red versus green fluorescence which is visualized in the left hand panel on the screen. The axis is then moved in order to gate out cells depending on the threshold. As the nuclei of interest are selected manually in the images, adjusting the gating thresholds above zero is not required. All pixel intensity values observed within the nuclear perimeters are considered as positive signal.</li>
	</ol>
	</li>
	<li>Repeat this process by selecting the third icon in the tab and encircling subsequent nuclei until the dataset is completed.</li>
	<li>To export this data, right click on the table, select save table and save to an appropriate location.</li>
	<li>To save the colocalization image, select the File menu and select &#39;Export&#8230;&#39; then choose; Tagged Image File (Format) and &#39;Contents of Image Window &#8211; single plane (Data)&#39; followed by clicking &#39;Select file name and save&#8230;&#39;, choose an appropriate location and click &#39;Save&#39;.</li>
	<li>Plot colocalization data in a spreadsheet followed by statistical analysis.</li>
</ol>

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No potential conflicts of interest were disclosed.

This protocol describes the stepwise process of generating visual representations of DNA modifications within nuclei. Whilst sensitive and impactful, these techniques do possess a number of limitations.
It is necessary to emphasize that the data generated from 2.5D plots, signal intensity profiles and colocalization analysis are semi-quantitative in nature. Owing to the point by point illumination of the sample during image acquisition on the laser scanning confocal microscope, absolute signal intensities of each channel are recorded. However, these are not absolute magnitudes of 5hmC and 5caC being detected and only represent indications of the presence, absence or scale of these epigenetic marks.
Due to the repetitive nature of signal amplifications, limitations consistent with the magnitude of machinery applied to enhance the signal inevitably confound approximations of true physical genomic occupancy of these marks34. The true genomic localization of these marks may be obscured by these bulky proteins, which physically occupy areas unresolvable even at optimal confocal resolution limits of 200-300 nm34. Moreover, the signal intensities of the individual channels for each mark cannot be compared to each other due to differences in antibody sensitivity and fluorophore conjugation34. However the information obtained with imaging sheds light on the spatial and temporal organization of the genomic marks.
For accurate quantitation of 5hmC and 5caC signals, nuclei are highlighted and encircled against the DAPI counterstain, clearly demarcating the region to be analyzed. This procedure dispenses with the requirement for calibrating gating thresholds as background noise is disregarded through the manual process of selecting nuclei1. An automation of this process can be achieved in the latest software which allows for nuclear segmentation to be performed. Whilst free alternative software packages such as FIJI are available for colocalization analysis, drawbacks such as the requirement to convert images to binary 8 bit formats, masking images and inputting size exclusion data to select regions of interest limit the user-accessibility of this program. Additionally, considering the overall limited levels of 5hmC and 5caC in the genome coupled with their predominant location at euchromatic regions and, depletion of genomic CpG sequences in general, manual encircling of nuclei introduces user-bias. Therefore, the highlighting of nuclei automatically assumes all events occurring within the demarcated region to be positive and disregards any background pixels which may be present. Thus, analyzing images based on pixel intensity may be more meaningful. These factors are important when considering the use of Mander&#39;s colocalization coefficient to analyse the degree of spatial overlap between two signals, irrespective of their signal intensities as oppose to Pearson&#39;s correlation coefficient which assumes a linear relationship between signals.
Overall, the techniques outlined here carry the theme of visual representation of biological data in an intuitive format. Whilst semi-quantitative image analysis cannot supersede powerful techniques such as mass spectrometry in terms of sensitivity or single base resolution genome wide sequencing, it does provide complementary data allowing inferences and hypotheses to be conceived from analysis of images with precision.

DNA methylation, a well-documented mechanism associated with transcriptional regulation entails modification of cytosine residues in a 5&#39;-cytosine-phosphate-guanine-3&#39; (CpG) dinucleotide context via the addition of a methyl group (-CH3) to the 5- carbon atom of the cytosine pyrimidine ring to form 5-methylcytosine (5mC)1. In mammals, approximately 70% of CpGs are methylated which constitutes only 1% of their genomes as they are depleted of this palindromic sequence owing to 5mC mutagenic propensity to spontaneously deaminate to thymine2. Presence of methyl groups on gene promoter sequences show strong correlation with transcriptional repression in vertebrates3,4,5. Addition of these methyl groups is catalyzed by highly conserved DNA methyltransferase (DNMT) enzymes DNMT3a, 3b and 3L, and DNMT1 which modify CpG cytosines in de novo and maintenance methylation contexts respectively6. DNMT3A/B expression is elevated during development in embryonic stem cells and epiblast; however its diminished expression is observed following pluripotent cell lineage commitment to somatic fates during differentiation8. Whilst sharing functional redundancy, DNMT3a and 3b display tissue-specific expression patterns, with 3a detected uniformly in mouse embryos but 3b predominantly localized to neuroectoderm and chorionic ectoderm tissues8.
Methylation signatures can be inherited during mitosis and meiosis10. Maintenance methylation involves DNMT1 facilitated modification of CpG cytosine residues existing in hemi-methylated palindromes on double-stranded DNA11. DNMT1 binds DNA at replication forks11 and consequently, genomic methylation levels peak during S phase of the cell cycle12. DNMT1 methylates unmodified cytosines thereby distinguishing newly synthesized DNA strands, promoting X-chromosome inactivation and maintaining transcriptional repression profiles13. Through recognition of hemi-methylated DNA CpG sequences, DNMT1 maintains established patterns of methylation demarcated by de novo methylation e.g. repression of Long Interspersed Nuclear Element 1 (LINE1) retrotransposon promoters to inhibit its potentially carcinogenic propagation14. Although possessing hemi-methylated DNA preferential binding affinity, DNMT1 can methylate unmethylated CpG island (CGI) sequences in DNMT3a/b -/- mutant cells, fulfilling an emergency de novo methylation role15. Thus owing to the semi-conservative nature of DNA replication16, maintenance methylation can faithfully recapitulate methylation signatures from parent to daughter cell17.
However, for gene expression to be dynamically modulated, repressive methylation modification must be erased, which can be achieved by passive and active demethylation mechanisms18. The Ten-Eleven Translocase (TET) proteins belonging to a conserved family of dioxygenase proteins are capable of iterative oxidation of methyl groups on CpG residues19. These Tet proteins, homologous to J-Base binding proteins (JBP) discovered in Trypanosome bruceii, recognise and bind modified DNA bases e.g. 5mC and oxygenate these residues to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)20. Tet protein facilitated oxidation of 5mC results in stepwise conformation change from methyl to hydroxyl, carbonyl and carboxylate configurations, however 5fC and 5caC modifications can be synthesized directly from 5mC oxidation21,22,23,24.
An informative indication of TET protein activity in understanding their regulation is studying the distribution and abundance of oxi-methyl-cytosine marks. Significant 5mC presence at CpG poor promoters is detectable in contrast to unmethylated CpG rich regions, the latter being characteristic of CpG islands25. Across tissues, highest tissue specific methylation is observed in brain, testis and blood whilst oral mucosa exhibit greatest hypomethylation, indicating a differential methylation pattern occurring at tissue specific promoters26.
Through utilizing sensitive anti-5mC and anti-5hmC antibodies in selective methyl/hydroxymethyl DNA immunoprecipitation (meDIP/hmeDIP), and subsequent high throughput sequencing, Ficz et al. demonstrated high 5hmC occupancy at promoters, exons and LINE-1 retrotransposon sequences which correlated with reduced 5mC levels at these locations in mouse embryonic stem cells27. Inversely, greatest 5mC enrichment was observed at repetitive satellite sequences where 5hmC presence was limited28. Studies performed on human frontal lobe brain tissue reveal highly significant 5hmC enrichment, four fold higher than in mouse embryonic stem cells28. In concordance with previous observations, high throughput sequencing of frontal lobe tissue illustrated majority 55-59% of 5hmC signal localized at low density CpG promoter regions, 35-38% within gene bodies and approximately 6% occupancy at intergenic regions. In contrast, 5mC was enriched at intergenic regions (25-26%) and higher within gene bodies (52-55%) but reduced (22-24%) at promoter sequences29. These studies indicate abundance of 5hmC in embryonic stem cells and somatic tissue, particularly the brain, however investigations on 5fC and 5caC distributions are limited.
Interestingly, recently discovered in eukaryotes, methylation of adenine residues at the position 6 nitrogen (N6) (6mA) display a genomic abundance profile inverse to that of 5mC30. Observations from liquid chromatography coupled tandem mass spectrometry reveal 6mA absolute levels to exist in excess in zebra fish and porcine early embryos compared to sperm with its levels (0.003% of genomic adenines) increasing steadily upon fertilization, peaking at the morula developmental stage (33 fold higher than sperm) and reaching steady state somatic levels of 0.004% of genomic adenines31. Immunoprecipitation of 6mA enriched DNA sequences has demonstrated predominant occupancy (approximately 80% enrichment) of this mark at repetitive element regions and transcriptional start sites32. These observations contextualize and validate the discovery of 6mA demethylase-null embryonic stem cells exhibiting accumulated 6mA mediated LINE-1 retrotransposon silencing compared to transcriptionally active elements in wildtype cells. These data suggest a transcriptional regulatory function for 6mA33.
Whilst conjugated biochemical tags coupled to subsequent DIP assays indicate presence or absence of oxidized methylcytosine derivatives (oxi-mCs), they cannot impart spatial distribution or quantifiable information of these marks34,35. A protocol for sensitive immunochemical detection of 5hmC and 5caC was recently developed36. This fluorophore conjugated secondary antibody-based immunostaining method coupled with utilizing scanning laser confocal microscopy possesses the unique advantage of providing visual localization of these DNA modifications within the cells, thus, emphasizing individual positively or negatively stained cells corresponding with the heterogeneous presence of these marks. The 5hmC and 5caC absolute signal intensities as amplified by the conjugated antibodies enable semi-quantitative interpretations to be proposed about the magnitude and positions of these marks within the nucleus e.g. heterochromatic and euchromatic regions37,38. Here a technique for the computational analysis of confocal microscopy images is described. The generation of 2.5D spatial distribution plots for displaying distinct 5hmC and 5caC signal peaks per pixel and their locations within nuclei is demonstrated. Histogram plots of 5hmC and 5caC signal intensity profiles can illustrate trends in abundance of these marks as the peaks and troughs are plotted as separate non-overlapping channels. Finally, by implementing the colocalization function, the degree of proximity of one signal to another can be determined and as a result of this, their respective genomic coordinates can be identified.

<table><tbody><tr><td>Zeiss Elyra PS1. Super resolution microscope equipped with LSM780 confocal head</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>Zeiss Zen Black 2012</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>Zen Blue 2012</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>NA</td><td></td></tr></tbody></table>

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-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.

To determine the spatial distribution of 5hmC and 5caC in differentiating hepatic progenitors immunostained slides for these DNA modifications were imaged using a microscope.
Initial analysis of the spatial distribution of these oxi-mCs was carried out through the generation of 2.5D intensity plots (Figure 1A, 1B). The red and green peaks appeared to be well defined with the limited presence of orange peaks indicating only a small overlap of the 5caC and 5hmC signals. In agreement with these results, the profiles for 5hmC and 5caC intensities in the corresponding cell do not strongly coincide with each other (Figure 1C). This illustrates that 5caC and 5hmC exhibit distinct patterns of nuclear distribution in hepatic progenitors suggesting that Tet-dependent oxidation of 5mC leads to generation of different oxidized derivatives of this DNA modification in specific chromatin regions.
To compare the levels of 5caC and 5hmC in Daoy medulloblastoma and BXD-1425EPN ependymoma cells, immunostaining for these oxi-mCs was carried out on both samples under identical experimental conditions. Intensities of 5caC and 5hmC signals were then compared in 20 profiles generated across 3-5 cells recorded for each cell line (Figure 2A-2D). The quantification of these results revealed that BXD-1425EPN cells exhibit significantly lower levels of both 5hmC and 5caC immunostaining compared to Daoy cells (Figure 2D).
Considering the two channels red (5hmC) and green (5caC) which can be denoted as R and G respectively, the Pearson&#39;s correlation coefficient (PCC) describes the degree of spatial overlap and co-segregation of R &#38; G assuming both marks exist in a linear relationship with each other, denoted by R statistic34. Squaring the PCC value, denoted by R2 enables the measurement of green signal intensity variation which is influenced by red signal intensity34. This is superfluous for our 5caC vs 5hmC co-localization analysis.
To examine the spatial distribution of 5caC and 5hmC in undifferentiated (Undiff) hiPSCs and hepatic endoderm 24 hours (HE24) after induction, colocalization analysis of these DNA modifications was carried out on the same confocal images, with R values for 20 nuclei analysed for each cell line (Figure 3A-3C).
The quantification of these results demonstrated that degree of colocalization is significantly higher (P &#60; 0.05) in HE24 compared to Undiff (Figure 3c). This may be attributable to the reduced magnitude of 5caC presence and thus its reduced signal intensity in Undiff cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56318/56318fig1.jpg" /> 
Figure 1: Immunohistochemical staining of hepatic endoderm progenitors at 72 hours post induction of differentiation. (A) Co-staining of 5hmC (red), 5caC (green) and DAPI nuclear counter stain (blue). The white dashed arrow &#39;B&#39; denotes the direction of profiling sampled through the nucleus, illustrated in 1C. Scale bar represents 5 &#181;m. Red channel: gain 800, laser 1%. Green channel: gain 750, laser 2.4%. DAPI channel: gain 750, laser 2.6%. (B) 2.5D intensity plot of 5hmC, 5caC and DAPI signals. Individual peaks represent absolute signal intensities of each pixel. Merged views and individual channels are shown. (C) The intensity profiles of 5hmC, 5caC and DAPI signals in hepatic progenitors at 72 hours post induction of differentiation. Peak intensities were recorded along the dimensions of the white dashed arrow &#39;B&#39; and are indicated along the intensity profile x-axis by a black dashed arrow. <a href="https://cloudflare.jove.com/files/ftp_upload/56318/56318fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56318/56318fig2.jpg" /> 
Figure 2: Analysis of 5hmC and 5caC signal intensities in Daoy medulloblastoma and BXD-1425EPN ependymoma cell lines. (A) Immunofluorescent staining of 5hmC (red) and 5caC (green) in Daoy and BXD-1425EPN cells. Images were captured with 63x oil immersion objective lens, both at the same settings. Merged views and individual channels are shown. Scale bars represent 10 &#181;m. Red channel: gain 746, laser 1%. Green channel: gain 750, laser 2.4%. Individual (B) Daoy and (C) BXD-1425EPN cells are expanded in adjacent figures. Scale bars for (B) and (C) represent 5 &#181;m. (D) Mean fluorescence intensity of 5hmC vs 5caC signals in Daoy and BXD-1425EPN cells (n = 20, SD). Significance was assessed using an F-test and Two-sample t-Test. Asterisks denote statistically significant p-values of ** as p &#60;0.01 and *** as p &#60;0.001. <a href="https://cloudflare.jove.com/files/ftp_upload/56318/56318fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56318/56318fig3.jpg" /> 
Figure 3: Colocalization analysis of 5hmC and 5caC absolute signals in undifferentiated hiPSCs (Undiff) compared to hepatic endoderm 24 hours post-differentiated progenitor cells (HE24). Confocal microscopy images of (A) undifferentiated hiPSCs and (B) HE24 cells stained for 5hmC (red), 5caC (green) and DAPI (blue). Cells were imaged with 63X oil immersion lens at identical settings. 20 encircled nuclei illustrate the cells sampled for colocalization analysis. Scale bars represent 20 &#181;m. Red channel: gain 800, laser 1%. Green channel: gain 750, laser 2.4%. DAPI channel: gain 750, laser 2.6%. (C) Colocalization analysis of the presence of 5caC signal within range of 5hmC proximity in undiff and HE24 (n = 20, SD). Significance was assessed using an F-test and Two-sample t-Test. Asterisks denote statistically significant p-values of * p &#60;0.05. <a href="https://cloudflare.jove.com/files/ftp_upload/56318/56318fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Immunostaining for DNA Modifications: Computational Analysis of Confocal Images at JoVE.com

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

immunostaining-for-dna-modifications-computational-analysis-confocal

Research

JoVE Journal

Genetics

9.6K Views.  University of Nottingham. Onlangs ontdekte geoxideerde vormen van 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) en 5-carboxylcytosine (5caC) kan verschillende DNA wijzigingen met unieke functionele rollen. Hier wordt een semi-kwantitatieve workflow voor visualisatie van oxi-mCs ruimtelijke verdeling, signaal intensiteit profilering en colocalization omschreven.

Video: Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

Immunology and Infection

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Cancer Research

Analysis Workflow of Nuclear Protein Staining in Human Primary Cells

A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience.
In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution.
Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different&#160;contexts.

Author Spotlight: Comprehensive Epigenetic Analysis for Investigating Human Cellular Plasticity and Environmental Adaptation Using Immunofluorescence Assays

This method describes an immunofluorescence protocol and quantification pipeline for evaluating protein distribution with varied nuclear organization patterns in human T lymphocytes. This protocol provides step-by-step guidance, starting from sample preparation and continuing through the execution of semi-automated analysis in Fiji, concluding with data handling by a Google Colab notebook.

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the ...

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Developmental Biology

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Biology

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

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