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Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry
- Source :
- Methods in Molecular Biology ISBN: 9781071608753
- Publication Year :
- 2020
- Publisher :
- Springer US, 2020.
-
Abstract
- 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.
- Subjects :
- 0303 health sciences
biology
medicine.diagnostic_test
Chemistry
Immunocytochemistry
Colocalization
Immunofluorescence
Primary and secondary antibodies
Cell biology
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
030220 oncology & carcinogenesis
biology.protein
medicine
Immunohistochemistry
Epigenetics
DNA
Immunostaining
030304 developmental biology
Subjects
Details
- ISBN :
- 978-1-07-160875-3
- ISBNs :
- 9781071608753
- Database :
- OpenAIRE
- Journal :
- Methods in Molecular Biology ISBN: 9781071608753
- Accession number :
- edsair.doi...........90708671d8cf96601c9e435f62746056