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Already, the two-dimensional (2D) analysis of FISH and immunofluorescence data reveal the location of chromosomes as well as the different markings on the spermatic nucleus. In addition, a good 3D rendering after Imaris software processing was obtained when Z-stacks of images were acquired over a defined volume (10 μm × 13 μm × 15 μm) with a sequential scanning mode to minimize bleed-through effects and avoid overlapping wavelengths.Computational analysis of digital images provides a robust and unbiased way to compare and investigate the amount (pixel intensity) and spatial distribution of DNA modifications. The DNA modifications in the cells are visualized by fluorescence labeling and the images are captured by confocal microscopy. The key advantage of the confocal over conventional microscope is that it images only a thin optical section around the focal plane of the microscope therefore it can precisely record signals only from the focal plane inside the nucleus. In this chapter, we will describe in detail several analysis methods to visualize and quantify the DNA modification signals including how to investigate codistribution of such signals when using dual labeling.Immunostaining (also called as immunofluorescence) is a fluorescence labeling method to stain one or more epitopes of interest on DNA and/or protein using specific antibodies. Cytosine modifications can be detected quantitatively by immunostaining. The protocol commonly includes sequential steps. These include fixation, permeabilization, antigen retrieval, blocking, incubation with primary and secondary antibodies, and visualization under the microscope followed by image-based intensity analysis of staining. Each step is important, but antigen retrieval is especially necessary for DNA epitopes such as cytosine modifications as antibodies can access cytosines in DNA only once the DNA double-strand is denatured and DNA-packaging proteins have been removed. Hydrochloric acid is commonly used for this purpose. https://www.selleckchem.com/products/Decitabine.html However, there are additional treatments with enzymes to enhance antigen retrieval and improve the detection by increasing staining intensity. This chapter describes current methodology for improving antigen retrieval for the staining of the cytosine modifications 5'-methylcytosine (5meC), 5'-hydroxymethylcytosine (5hmC), 5'-formylcytosine (5fC), and 5'-carboxycytosine (5caC).Methylated cytosine (5-methylcytosine) is the most studied epigenetic mark involved in the regulation of gene expression. Although it displays highly variable dynamics during plant ontogenesis, it is possible to gain a fine spatial perspective with immunohistochemistry techniques that use specific antibodies and fluorochromes. Besides, there are other cytosine modifications described in plants, although their biological significance is still unknown (i.e., 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine). Here we present a standardized protocol to detect cytosine modifications in plant tissues.5-methylcytosine (5mC) is an epigenetic modification to DNA which modulates transcription. 5mC can be sequentially oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Collectively, these marks are referred to as the oxidized derivatives of 5mC (i.e., oxi-mCs). Their formation is catalyzed by the ten-eleven translocation methylcytosine dioxygenases (TETs 1, 2 and 3). Various techniques have been developed for the detection of oxi-mCs. The following chapter describes an immunochemical protocol for the simultaneous detection of 5hmC and 5caC in embryonic zebrafish tissue sections. The embryos are fixed, permeabilized and embedded in paraffin blocks. The blocks are cut into sections that are mounted onto slides. Depurination of the DNA is performed to allow immunodetection of the oxi-mCs. The 5hmC is detected with the help of a mouse anti-5hmC monoclonal primary antibody and a goat anti-mouse Alexa Fluor 633-conjugated secondary antibody. The weak 5caC signal requires enzymatic amplification. Its detection involves a rabbit anti-5caC polyclonal primary antibody and a goat anti-rabbit secondary antibody that is conjugated to horseradish peroxidase (HRP). HRP amplifies the 5caC signal by catalyzing the deposition of large quantities of fluorescein-labeled tyramide. Sections immunostained for 5hmC and 5caC are analyzed by fluorescent light or confocal laser scanning microscopy. This immunochemical method allows for highly sensitive detection of 5hmC and 5caC in zebrafish tissues.The modified cytosine base 5-hydroxymethylcytosine (5hmC) is abundantly present in the central nervous system (CNS), and visualization of global 5hmC levels is possible through use of immunohistochemistry. In this chapter we describe an adaptable method of brain tissue collection and immunohistochemical staining that allows for detection of 5hmC in mouse or rat brain, meaning that the method can be applied to many rodent models of CNS diseases and disorders.Immunocytochemistry can be instrumental in assessing the spatial distribution and relative levels of epigenetic modifications. Although conventional immunostaining has been utilized for the detection of 5-methylcytosine (5mC) in animal cells and tissues for several decades, the sensitivity of techniques based on the use of fluorophore-conjugated secondary antibodies is not always sufficient for studying DNA modifications that are less abundant in DNA compared with 5mC. Here we describe a protocol for sensitive immunocytochemistry that utilizes peroxidase-conjugated secondary antibodies coupled with catalyzed reporter deposition and allows for detection of low-abundance noncanonical bases (e.g., 5-carboxylcytosine, 5caC, 5-formylcytosine, 5fC, 5-hydroxymethyluracil, 5hmU) in mammalian DNA. This method can be employed for evaluation of the levels and nuclear distribution of DNA modifications and permits their colocalization with protein markers in animal cells.The lampbrush chromosomes found in the giant nucleus or germinal vesicle (GV) of amphibian oocytes provide unique opportunities for discrete closed and open chromatin structural domains to be directly observable by simple light microscopy. Moreover, the method described here for preparing spreads of lampbrush chromatin for immunostaining enables a straightforward approach to establishing the distributions of modified nucleotides within and between structurally and functionally distinctive chromatin domains.DNA methylation undergoes dynamic changes at the genome-wide scale during the early steps of mammalian embryo development. link2 Immunochemical detection of 5-methylcytosine (5mC) in the zygote has led to the discovery that a global loss of DNA methylation takes place soon after fertilization, occurring rapidly in the paternal pronucleus. Using the same method employed above, which detects modified bases in the denatured single stranded DNA, we showed that this active DNA "demethylation" in the paternal pronucleus involves oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) by the TET3 enzyme. By immunostaining of genetically altered zygotes we revealed that the maternal pronucleus is protected from TET3-mediated oxidation by histone H3K9 methyltransferase enzymes, EHMT2 and SETDB1. The same assays are also applicable for visualizing the temporal and spatial distribution of the modified cytosine residues in preimplantation embryos. Here, we provide a detailed protocol for detecting 5mC, 5hmC, 5fC, and 5caC in mouse zygotes and preimplantation-stage embryos using antibodies raised against modified cytosine species.The oxidation activity of the mammalian ten-eleven translocation dioxygenase (TET) on 5-methylcytosine (5mC) of DNA is usually monitored by analytical methods such as dot blotting and liquid chromatography-mass spectrometry (LC-MS). Herein, we describe a high throughput capillary gel electrophoresis assay for monitoring the in vitro oxidation of 5mC by TET. The method is rapid and quantitative, and can serve as a powerful tool in mechanistic studies of TET.Reliable quantitative analysis of DNA modification using liquid chromatography coupled with tandem mass spectrometry requires stable isotope-labeled internal standards. Only some of them are commercially available. Here we present a method allowing for the synthesis of [13C10,15N2]-5-methyl-2'-deoxycytidine from [13C10,15N2]-2'-deoxythymidine. We also describe an approach for the oxidation of [13C10,15N2]-5-methyl-2'-deoxycytidine and [13C10,15N2]-2'-deoxythymidine with Na2S2O8, leading to the generation of [13C10,15N2]-5-formyl-2'-deoxycytidine, [13C10,15N2]-5-carboxy-2'-deoxycytidine or [13C10,15N2]-5-(hydroxymethyl)-2'-deoxyuridine, correspondingly. Moreover, we provide optimized protocols for the oxidation of [13C5,15N2]-thymine to [13C10,15N2]-5-hydroxymethyluracil, [13C10,15N2]-5-formyluracil, and [13C10,15N2]-5-carboxyuracil using Na2S2O8.Analytical techniques based on mass spectrometry allow to analyze DNA modifications in body fluids. Here we describe two chromatographic methods that can be used for the simultaneous determination of the modified DNA bases and nucleosides in the same urine sample isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) and high-performance liquid chromatography coupled with gas chromatography and mass spectrometry (HPLC/GC/MS).Our hereby presented methodology is suitable for reliable assessment of the most common DNA modifications which arise as a product of fundamental metabolic processes. 8-oxoguanine, one of the oxidatively modified DNA bases is a typical biomarker of oxidative stress. A noncanonical base, uracil, may also be present in small quantities in DNA. Ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can also be generated by TET enzymes. All the above mentioned modifications seem to play some regulatory roles. Here, we provide a protocol for isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. We also provide optimized protocols for extraction of DNA, fully compatible with the downstream MS/MS analysis.DNA methylation serves to mark DNA as either a directed epigenetic signaling modification or in response to DNA lesions. Methods for detecting DNA methylation have become increasingly more specific and sensitive over time. Conventional methods for detecting DNA methylation, ranging from paper chromatography to differential restriction enzyme digestion preference to dot blots, have more recently been supplemented by ultrahigh performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) to accurately quantify specific DNA methylation. Methylated DNA can also be sequenced by either methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) or single-molecule real-time sequencing (SMRTseq) for identifying genomic locations of DNA methylation. link3 Here we describe a protocol for the detection and quantification of epigenetic signaling DNA methylation modifications including, N6-methyladenine (6mA), N4-methylcytosine (4mC) and C5-methylcytosine (5mC) in genomic DNA by triple quadrupole liquid chromatography coupled with tandem mass spectrometry (QQQ-LC-MS/MS).
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