Another successfully-tested, modified nucleoside was bis(POM)-phosphorylated EdU (PEdU), that was also less toxic than EdU . Although all the aforementioned nucleoside analogs exhibit lower toxicity than EdU according to the authors, their use as compared to EdU or BrdU is very limited. the immunochemical detection of the incorporated BrdU was described in denaturated DNA . Four years later, Brevianamide F the method of the immunofluorescence in in situ detection of incorporated BrdU in nuclear DNA was developed . Besides the immuno-detection of BrdU, the detection system based on the finding that the fluorescence of Hoechst stains bound to DNA Brevianamide F is suppressed by incorporated BrdU was used . Although this system does not require any special DNA treatment, it is not Brevianamide F suitable for common microscopic studies. On the other hand, it was successfully used for the analysis of the cell cycle using flow cytometry [34,35,36]. As BrdU and also other halogen derivatives of thymidine are inaccessible in double-stranded DNA for reactions with antibodies, it is necessary to use special treatments to make them accessible for such reactions. In this respect, the protocols based on the use of acid or hydroxide treatments are very common approaches for BrdU detection [37,38,39,40,41]. While the hydrochloric-acid solutions results in depurination and cleavage of the DNA, the treatment with the sodium hydroxide results in a loosening of the DNA structure as a consequence of the deprotonation of the nucleobases . The use of strong acids also leads to the partial destruction of cellular components. For example, the use of highly-concentrated HCl (usually 1.5 MC4 M) leads to the destruction of many nuclear antigens [38,40,42,43]. In the case of the concurrent labeling of cellular DNA by DNA dyes such as DAPI, Hoechst dyes, or propidium iodide, such treatment can substantially decrease their signal [42,44]. Protocols based on the use of strong alkalis, although commonly used in lower concentrations, exhibit a similar negative impact on the cellular structures [40,42]. The above-mentioned problems led to the development of alternative approaches of BrdU detection. Brevianamide F One common alternative method is based on the cleavage of DNA by nucleases. Either a type of nuclease, or a mixture of various nucleases, is used to generate short single-stranded tracts accessible for binding the anti-BrdU antibodies [38,40,45,46]. The use of nucleases is one of the approaches that does not exhibit a strong negative effect on the cellular structure. The approach based on nucleases was further optimized from the viewpoint of its effectivity, and currently it is one of the best methods for the detection of DNA replication, even if a short labeling pulse of BrdU is used (Figure 1), enabling co-localization studies including those with fluorescent proteins as well . Open in a separate window Figure 1 The detection of BrdU using DNase I and exonuclease Nrp1 III. Examples of microscopic images of cell nuclei labeled for 5 min with BrdU and fixed with formaldehyde. The DNA was labeled by DAPI. The scale bar50 m. Adapted with permission from Ligasov et al., 2017, Looking for ugly ducklings: The role of the stability of the BrdU-antibody complex and the improved method of the detection of DNA replication. PLoS ONE 12(3): e0174893 . A simple and very efficient method of BrdU detection providing high signal/background ratio is based on the use of low concentration of HCl and exonuclease III . In this case, the breaks in the DNA are introduced by the low concentration of hydrochloric acid followed by the subsequent enzymatic extension of these breaks using exonuclease III. This approach enables, at least to some extent, co-localization studies with fluorescent proteins. Concerning the cellular proteins, the immunocytochemical detection of proteins can vary from protein to protein and from antibody to antibody; therefore, the impact of the method on the localization and signal intensity of particular protein should be tested first . Another alternative for the detection of BrdU is the cleavage of DNA using monovalent copper ions. This method is based on the oxidative attack at the deoxyribose moiety by monovalent copper in the presence of oxygen Brevianamide F . This approach provides a high signal/background ratio, is quickly feasible, does not require any special equipment, and preserves at least some type of protein antigens . A further method of BrdU detection is based on the photolysis of BrdU-labeled DNA followed by the detection of the induced breaks . This method uses illumination of cells with UV light to selectively photolyze DNA with the incorporated BrdU [36,49,50]. Heat denaturation is another way.
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