Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge

Çelik-Uzuner, Selcen

doi:10.1016/j.jim.2014.11.011

Cited by 5 publications

(5 citation statements)

References 181 publications

(227 reference statements)

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“…Antibody‐based determination of cytosine modifications by fluorescence microscopy has been commonly used, as it is advantageous for the simultaneous specific and selective analyses of modifications. There are different antigen retrieval strategies, and hydrochloric acid (HCl) is the most common chemical used for the staining of cytosine modifications (Celik, ). The application of acid can vary according to a range of conditions, including concentration and incubation.…”

Section: Discussionmentioning

confidence: 99%

“…There are different antigen retrieval strategies, and hydrochloric acid (HCl) is the most common chemical used for the staining of cytosine modifications (Celik, 2015). The application of acid can vary according to a range of conditions, including concentration and incubation.…”

Section: Discussionmentioning

confidence: 99%

“…There are many types of methodologies for the detection of cytosine modifications at the gene or genome level. Antibody‐based applications using specific antibodies have been commonly performed for the detection of genomic level of modifications by fluorescence‐based instruments such as flow cytometers, ELISA‐plate readers, and fluorescence microscopy (Celik, ; Kurdyukov & Bullock, ). ELISA‐based measurements require isolated DNA; however, flow cytometry and fluorescence microscopy require fixed cells for the detection of levels of cytosine modifications.…”

Section: Introductionmentioning

confidence: 99%

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Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Çelik-Uzuner

2019

Microsc Res Tech

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Epigenetic modifications of DNA, including methylation, hydroxymethylation, formylation, and carboxylation of cytosines, are proposed to function in gene regulation during reproduction and development. Changes in cytosine methylation are associated with a range of diseases, such as cancer. Immunofluorescence uses specific antibodies to quantitatively detect the global amount of cytosine modifications by fluorescence microscopy. The most critical stage of immunofluorescence is the antigen retrieval to remove the protein content around the DNA, allowing specific antibodies to bind to DNA epitopes. Acid treatments have commonly been used for antigen retrieval. Previously, trypsin was added after acid in the protocol, which increased the amount of detectable DNA methylation. In this study, the protocol was further enhanced by the addition of pepsin, which is able to target charged hydrophobic amino acids in proteins, unlike trypsin, which breaks positive hydrophilic amino acids. The global levels of cytosine modifications in CF‐1, HeLa, and AR42J cells were compared using this protocol. In all cells, the sequential treatment of trypsin and pepsin increased the specificity of the staining. With the synergistic effect of the two enzymes, it is possible to target different protein groups packaging DNA molecules and removing them effectively. The findings suggest that this revised protocol can be conveniently used for each cytosine modification in the cells examined, and should be optimized for other cells. These new antigen retrieval conditions may more accurately detect the changes in cytosine modifications during development and in diseases.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Çelik-Uzuner

2019

Microsc Res Tech

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“…In AD, however, it remains controversial whether epigenetic marks change in affected brain regions or if changes merely reflect age related epigenetic alterations. It was the aim of this study to measure these epigenetic marks using a more specific technique, mass spectrometry, instead of the semi‐quantitative method of immunochemistry where antigen retrieval and non‐specific antibody binding have the potential to cloud results (Yuan ; Celik ). To determine epigenetic changes in brain regions associated with AD throughout the progression of the disease, modifications to cytosine were analyzed using GC/MS and stable labeled standards of each cytosine base (Fig.…”

Section: Discussionmentioning

confidence: 99%

Multiregional analysis of global 5‐methylcytosine and 5‐hydroxymethylcytosine throughout the progression of Alzheimer's disease

Ellison

Abner

Lovell

2017

Journal of Neurochemistry

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Epigenetic modifications to cytosine are known to alter transcriptional states and deregulate gene expression in cancer, embryonic development, and most recently in neurodegeneration. To test the hypothesis that global levels of cytosine modification are altered throughout the progression of Alzheimer’s disease, 5-methylcytosine (5-mC) and 5-hydroxmethylcytosine (5-hmC) were quantified using gas chromatography/mass spectrometry (GC/MS) and stable labeled internal standards of cytosine, 5-mC, and 5-hmC. Cytosine modifications were quantified in DNA extracted from tissue specimens of four brain regions (cerebellum, inferior parietal lobe, superior and middle temporal gyrus, and hippocampus/parahippocampal gyrus) of cognitively normal control (NC) subjects and subjects with mild cognitive impairment (MCI), preclinical Alzheimer’s disease (PCAD), late onset Alzheimer’s disease (LOAD), frontotemporal lobar degeneration (FTLD) and dementia with Lewy bodies (DLB). Repeated measures analyses of the data show significant alterations in 5-mC and 5-hmC in early stages of Alzheimer’s disease (PCAD and MCI), as well as FTLD and DLB subjects, across multiple regions of the brain. These data suggest alterations in epigenetic regulation of genes may play an early role in the progression of AD as well as other types of neurodegeneration.

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“…Careful design and validation of immunodetection of DNA methylation is required to optimizeoptimise epitope retrieval in each system under analysis. Conditions for testing in each system include technical conditions, such as the antibody incubation time and temperature required to achieve equilibrium binding conditions [37], the form and extent of antigen retrieval procedures [14][15][16]38,39], and the nature of the blocking protocol used to minimize non-specific binding and maximize, the form and extent of antigen retrieval procedures , and the nature of the two proteins that regulate methylation activity, UHRF1 and GADD45A, are also recruited to sites of repair and can catalyse selective methylation of the promoter-distal segment of the damaged DNA [34]. DNMT1 accumulation occurs in UV-induced lesions [35] and was detected in micronuclei in doxorubicin-treated cells [36].…”

Section: Discussionmentioning

confidence: 99%

The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts

Çelik-Uzuner

O’Neill

2015

Experimental Cell Research

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Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge

Cited by 5 publications

References 181 publications

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Multiregional analysis of global 5‐methylcytosine and 5‐hydroxymethylcytosine throughout the progression of Alzheimer's disease

The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts

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