ArticleHistochemistry and cell biology2023
Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications.
Article in Histochemistry and cell biology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed.
- Heat-induced antigen retrieval by using Tris-EDTA solution destroys nuclear structure in certain tissues.Biochemistry and biophysics reports · 2026Article
- EpiBrain: the brain's epigenetic landscape in a snapshot.bioRxiv : the preprint server for biology · 2026Article
- Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue.Journal of visualized experiments : JoVE · 2024Article
- Inhibition of cytosine 5-hydroxymethylation during progression of cancer precursor lesions in the uterine cervix.PloS one · 2024Article
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5 authors.
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Abstract
This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris-ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.
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