We thank the Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) and the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), the Institute of Neuroscience at the ULB and the FNRS for help and support. This work was supported by MRC (MR/L001047/1 to A.D.J.).

All the animal-involved procedures were performed in accordance with the University of Nottingham&#39;s ethical review board.
1. Selecting Suitable Tissue Preparation for Immunostaining
<ol>
	<li>Generate paraffin-embedded section of wild-type CD1 mouse embryos and adult brain tissues as described previously25. Use brain tissue sections fixed with either 4% formaldehyde (FA) or 4% paraformaldehyde (PFA) for immunostaining method25. 
	NOTE: The use of paraffin embedded tissue sections requires de-waxing prior to antibody labelling. Since the treatment with 2 - 4 M hydrochloric acid (HCl) employed in the protocol for DNA denaturing is not compatible with most antigen retrieval strategies, we recommend the use of either cryo- or microtome sections for co-detection of 5mC oxidation derivatives with protein markers.</li>
</ol>
2. Dewaxing Paraffin Embedded Tissue Sections
<ol>
	<li>In a running class II safety cabinet, wash the paraffin embedded tissue sections in a Coplin jar filled with xylene, 2 times for 10 min each at RT. 
	NOTE: It is important to use fresh xylene as incomplete paraffin removal can lead to inconsistent staining patterns.</li>
	<li>After de-waxing, rapidly rehydrate the tissue sections by washing consecutively in 95, 75, and 50% ethanol for 10 min each at RT.</li>
</ol>
3. Fixation and Permeabilization of Cryo- and Microtome Sections
<ol>
	<li>Fix the rehydrated tissue sections by placing them in either ice cold 4% PFA or 4% FA for 15 min at RT.</li>
	<li>Remove excess fixative by washing the sections in PBS (Phosphate buffer saline) for 5 min at RT.</li>
	<li>Permeabilize the tissue sections by placing in a Coplin jar filled with PBX (0.5% Triton X-100 in PBS) for 30 min at RT. Remove excess PBX by washing the sections shortly in PBT (0.01% Tween 20 in PBS).</li>
</ol>
4. Immunostaining for Oxi-5mC Derivatives
<ol>
	<li>Place the permeabilized-sections in 2 N HCl for 60 min at RT for depurination of the DNA. 
	NOTE: Although higher concentrations of HCl (e.g., 4 N) lead to a more efficient DNA denaturing, they are not compatible for co-detection of oxi-mCs with DAPI.</li>
	<li>Place the sections in 10 mM Tris-HCl (pH 8.5) for 30 min at RT to neutralize the HCl. Alternatively, wash the sections three times for 5 mins each in PBS. Incubate the sections in PBT for 5 min at RT.</li>
	<li>Carefully remove the liquid from the area surrounding the tissue section without letting the tissue section to dry completely at any step by gently shaking the slide. Use a hydrophobic barrier pen to encircle the section without touching the section. 
	NOTE: The hydrophobic barrier pen reduces the volume of antibody required to stain the tissue and can allow multiple sections to be stained with different antibodies on the same slide.</li>
	<li>Incubate sections in 100 &#181;l of blocking solution (10% bovine serum albumin in PBS) for 1 hr at RT in a humid chamber. 
	NOTE: Skipping this step would not affect the efficiency of staining the modified cytosine bases.</li>
	<li>Incubate the tissue sections in 100 &#181;l of a 1:5,000 dilution of mouse monoclonal anti-5hmC and a 1:1,000 dilution of rabbit polyclonal anti-5caC primary antibodies in blocking solution for 1 hr at RT in a humid chamber. Alternatively, perform the incubation overnight at 4 &#176;C if needed. 
	NOTE: Sections processed without primary antibodies can serve as suitable negative controls for the staining procedure. The 12.5 days post coitum murine embryonic brain sections enriched in 5caC, 5fC and 5hmC 20 can be used as positive control.</li>
	<li>Remove excess antibodies by washing the sections in a Coplin jar filled with PBT three times for 5 min each in a Coplin jar at RT. 
	NOTE: Increasing the volume of washing solutions can reduce background staining.</li>
	<li>Remove excess PBT and, if necessary, encircle the sections again with the hydrophobic barrier pen as PBT contain detergent that can weaken the hydrophobic barrier.</li>
	<li>Make a 1:400 dilution of goat anti-rabbit HRP-conjugated antibody and a 1:400 dilution of donkey anti-mouse 555-conjugated antibody in blocking solution.</li>
	<li>Incubate the tissue sections in 100 &#181;l of secondary antibody mixture from step 4.9 for 1 hr at RT in a humid chamber.</li>
	<li>Wash the tissue sections in a Coplin jar filled with PBT three times for 5 min each at RT.</li>
	<li>Place the tissue sections in 100 &#181;l of a 1:200 dilution of tyramide in the tyramide signal amplification buffer for 2 min at RT.</li>
	<li>Immediately, remove excess tyramide solution by washing the slides three times for 5 min each in PBT.</li>
	<li>Carefully remove excess PBT and immediately cover the sections with a drop of a Mounting Medium (see Materials List).</li>
	<li>Gently place a coverslip on the tissue sections and immediately seal the coverslip with nail polish.</li>
	<li>Store the tissue sections at 4 &#176;C for several hr before microscopic examination. Examine under a fluorescent microscope at 405, 488 and/or 555 nm (10 - 40X magnification).</li>
</ol>

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The authors declare no conflict of interest.

Although the reported low abundance of 5mC oxidation derivatives, 5fC and 5caC in some tissues would present significant limitations for a standard immunochemistry protocol, the incorporation of peroxidase-conjugated secondary antibodies allowed the detection of these cytosine modifications in fixed tissues and cells (Figure 2). However, the optimal incubation time with the tyramide solution should be optimized experimentally for each individual batch of tyramide signal amplification kit where a linear relationship between signal intensity and duration of tyramide based signal amplification is observed, for details refer to Almeida et al. 201226. In addition, the signal/background ratio can be significantly enhanced by carrying the washes in a Coplin jar to allow efficient removal of excess antibodies. Following incubation with tyramide solution, it is important to immediately stop the reaction by washing in PBT solution to decrease background staining. It is critical not to allow the sections to dry at any point during the procedure.
The efficiency of DNA depurination can be improved by carrying out the depurination reaction at 37 &#176;C. While using 4 N HCl instead of 2 N enhances the staining of the modified forms of 5mC using 4 N HCl for DNA depurination would not permit co-staining with DAPI, as it interacts exclusively with double stranded DNA.
Although this technique can provide robust semi-quantitative assessment of the modified forms of cytosine bases where detectable, it cannot be used for evaluating the absolute levels of 5mC or its oxidation derivatives. Therefore, we recommend the use of other complementary but quantitative approaches16, 19 -24. Since the discovery of 5mC oxidation derivatives in mammalian genomes, several approaches have been developed to study their biological roles16, 19-24. Although these approaches can be valuable in determining the absolute levels of 5mC oxidation derivatives, they do not provide information concerning their spatial distribution20, 26. We have successfully used the method described here to map the spatial distribution and localization of 5mC oxidation derivatives in different cell types of the developing and adult brain20
By revealing their spatial distribution20, this technique can be crucial to understanding the biological implications and the fate of oxidized derivatives of 5mC in various biological contexts where these modified derivatives of 5mC can be detected including cellular differentiation, development and disease. In addition, the method we describe here can give semi-quantitative assessments of the oxidized derivatives of 5mC in different tissues by assessing the kinetics of peroxidase reaction (which is proportional to the staining intensity) at different incubation times with tyramide26.

Methylation of cytosine bases in DNA (5mC) represents a major epigenetic mark found in vertebrates genomes associated with transcriptional silencing1. 5mC is being introduced and maintained by DNA methyltransferases2-5, and has been shown to play important roles in a number of biological processes including genomic imprinting, X-chromosome inactivation, cellular differentiation, and development3, 6. Consequently, the disruption of 5mC genomic patterns is associated with a number of diseases7, 8-11. Despite the progress in understanding 5mC role in development and disease, it is still remain largely unknown how this mark is being removed in developing and adult tissues. Several potential mechanisms of DNA demethylation have been recently proposed including active and passive demethylation mechanisms12, 13, 14, 15. Discovery of the products of 5mC sequential oxidation mediated by ten-eleven translocation enzymes (tet1/2/3) such as 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in eukaryotic DNA 16, 17, 18, 19 prompted speculations whether they may serve as intermediates of DNA demethylation processes or act as stable epigenetic marks in their own right13. While the demonstration that the component of base excision repair, thymine-DNA glycosylase (TDG) can bind and remove both 5fC and 5caC from DNA19, 20 suggests a role for the modified 5mC derivatives in an active DNA demethylation. Recent evidence showing that 5fC/5caC can modulate the rate of RNA II processivity points to potential involvement of these marks in transcriptional regulation29. Due to this potential biological importance of oxidized forms of 5mC, a range of biochemical and antibody based techniques have been employed for studying their genomic distribution and global content16, 19-24.
Given that most of the vertebrate organs consist of different cell types and that the distribution of modified cytosine bases is tissue and cell-type specific 16-18, 20, 23, 25-27, determining the spatial distribution of oxidized 5mC derivatives in different tissues becomes an important experimental task necessary for unveiling their biological functions. Most of the biochemical and antibody based approaches do not provide any spatial information regarding the distribution of the modified forms of 5mC in different tissue and cell-types. In contrast, immunochemistry based techniques can provide a rapid tool for assessing the spatial distribution and nuclear localization of 5mC28 and its oxidized derivatives20. That is said, the reported very low abundance of 5fC (20 in every 106 cytosine) and 5caC (3 in every 106 cytosine) in the mouse genome18 represents a significant challenge for standard immunochemistry.
Here, we describe a highly sensitive immunochemical method that provides robust and a rapid detection of oxidized form of cytosine in mammalian brain tissue. By incorporating peroxidase-conjugated secondary antibodies coupled with a signal amplification step, this method circumvents the challenges of detecting the very low amounts of 5fC and 5caC. In addition, this technique can be used to co-detect the modified forms of cytosine with lineage specific markers, effectively complementing other approaches in elucidating the biological functions of these epigenetic marks.

<table><tbody><tr><td>Coplin jar</td><td>Cole-Parmer</td><td>UY-48585-30</td><td>Glass made</td></tr><tr><td>Xylene</td><td></td><td></td><td>Use only in Class II safety cabinet</td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>Fisher Scientific</td><td>12821680</td><td>pH 7.5, filtered before use</td></tr><tr><td>4% paraformaldehyde (PFA)</td><td>Sigma Aldrich</td><td>P6148</td><td>Once made can be stored at -20 &amp;deg;C in aliquotes for several months</td></tr><tr><td>4% formaldehyde&amp;nbsp; (FA)</td><td>Sigma Aldrich</td><td>F8775</td><td>Once made can be stored at -20 &amp;deg;C in aliquotes for several months</td></tr><tr><td>Ethanol, anhydrous denatured, histological grade</td><td>Sigma Aldrich</td><td>64-17-5</td><td>95, 75, 50% in PBS</td></tr><tr><td>0.01% Tween 20 in PBS</td><td>Sigma Aldrich</td><td>P9416</td><td>PBT</td></tr><tr><td>0.5% Triton X-100 in PBS</td><td>Sigma Aldrich</td><td>X100</td><td>PBX</td></tr><tr><td>2 N HCl</td><td>Sigma Aldrich</td><td>71826</td><td>caution extremely toxic</td></tr><tr><td>100 mM Tris-HCl&amp;nbsp;</td><td>Promega</td><td>H5121</td><td>pH adjusted to 8.5</td></tr><tr><td>hydrophobic barrier pen</td><td>Abcam</td><td>ab2601</td><td>for immunohistochemistry</td></tr><tr><td>Slide moisture chamber</td><td>Scientific Device Laboratory</td><td>197-BL</td><td></td></tr><tr><td>anti-5mC mouse antibody</td><td>Diagenode</td><td>C15200081</td><td>monoclonal primary antibody</td></tr><tr><td>Anti-5hmC antibody</td><td>Active Motif</td><td>39791</td><td>rabbit polyclonal primary antibody</td></tr><tr><td>Anti-5caC antibody</td><td>Active Motif</td><td>61225</td><td>rabbit polyclonal primary antibody</td></tr><tr><td>Peroxidase-conjugated anti-rabbit seconday antibody</td><td>Dako</td><td>K1497</td><td></td></tr><tr><td>555-congjuated goat anti-mouse antibody</td><td>Molecular probes</td><td>A-11005</td><td>&amp;nbsp;secondary antibody</td></tr><tr><td>10% BSA</td><td>Sigma Aldrich</td><td>A9418</td><td>Blocking solution</td></tr><tr><td>22 x 32 mm Glass cover slips</td><td>BDH</td><td>406/0188/24</td><td></td></tr><tr><td>Tyramide Signal Amplification System</td><td>Perkin Elmer</td><td>NEL741001KT</td><td>Other fluorochome conjugated seconday antibodies can be used for co-detection of oxi-5mC&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Mounting Medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Colourless nail polish</td><td></td><td></td><td></td></tr><tr><td>chicken polyclonal anti-GFAP polyclonal antibody</td><td>Thermo Scientific</td><td>PA5-18598</td><td>1:400 dilution</td></tr><tr><td>anti-NeuN mouse monoclonal antibody</td><td>Merck milipore</td><td>MAB377B</td><td>1:400 dilution</td></tr></tbody></table>

detection of modified forms of cytosine using sensitive immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

To determine the distribution of 5hmC in brain tissue sections, we performed co-detection of this epigenetic modification with a marker for post-mitotic neurons, NeuN, employing commercial anti-5hmC antibody that specifically interacts with this mark but not with other forms of modified cytosine20, 25. Immunohistochemical analysis of 5hmC and 5caC distributions in the adult brain revealed that whereas the prominent 5hmC staining co-localizes with NeuN positive cells, NeuN-negative glial cells possess lower levels of genomic 5hmC (Figure 1)20.
We recently showed that Tet-dependent 5mC oxidation is operative during lineage specification of neural stem cells (NSCs) 20. Despite being immunochemically undetectable in NSCs, 5fC and 5caC exhibit prominent immunostaining at early stages of NSCs differentiation towards neuronal and glial lineages. Both these marks transiently accumulate concurrently with appearance of the markers of early neuronal and glial differentiation20. To determine the distribution of 5caC in differentiating NSCs we performed co-staining of this mark with a glial marker GFAP on the fixed cultures of NSCs at 3 days of induction of glial differentiation. Unlike in NSCs or mature astrocytes (data not shown), we observed strong 5caC signal in relatively large proportion of the cells expressing GFAP in these cultures (Figure 2)20.
<img alt="Figure 1" src="/files/ftp_upload/54416/54416fig1.jpg" /> 
Figure 1. Co-immunostaining of 5hmC (green) with NeuN (red) in the Adult Brain Tissue Counterstained with DAPI (Blue). Individual channels and the merged view are shown. Scale bars are 25 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54416/54416fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54416/54416fig2.jpg" /> 
Figure 2. Co-immunostaining of 5caC with GFAP in the Culture of NSC at Day 3 of Glial Differentiation. Individual channels and the merged view are shown. Scale bars are 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry at JoVE.com

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

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JoVE Journal

Biology

7.2K Views.  Université Libre de Bruxelles. Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Video: Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

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.

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 ...

Genetics

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Neuroscience

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Developmental Biology

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Biochemistry

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

Source: Jia, Z. et al., <a href="https://app.jove.com/v/55565">A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro.</a>&#160;J. Vis. Exp.&#160;(2017)
This video describes the technique of dot-blot to determine the level of DNA methylation in human chondrocytes in vitro via the blotting of DNA samples on a nylon membrane and the subsequent visualization using monoclonal antibodies. This helps to determine the chondrocytes&#39; phenotype at various stages in culture.

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

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Chapters in this video

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation