By Deryk T. Loo (auth.), Vladimir V. Didenko (eds.)
Recent advances in natural chemistry, fluorescent microscopy, and fabrics technology have created a wholly new variety of ideas and probes for imaging DNA harm in molecular and mobile biology. In DNA harm Detection In Situ, Ex Vivo, and In Vivo: equipment and Protocols, professional researchers discover the most recent advances within the region, masking either contemporary and validated thoughts to realize and quantify DNA harm at scales starting from subcellular to the extent of a complete reside organism. Chapters current all significant assays utilized in molecular and mobile biology for the labeling of DNA harm in situ, ex vivo, and in vivo. Composed within the hugely profitable equipment in Molecular Biology™ sequence structure, each one bankruptcy features a short advent, step by step equipment, an inventory of important fabrics, and a Notes part which stocks tips about troubleshooting and warding off identified pitfalls. accomplished and present, DNA harm Detection In Situ, Ex Vivo, and In Vivo: tools and Protocols is an important instruction manual for beginner and skilled researchers in various fields, together with molecular and mobile biology, experimental and scientific pathology, toxicology, radiobiology, oncology, embryology, experimental pharmacology, drug layout, and environmental science.
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Extra resources for DNA Damage Detection In Situ, Ex Vivo, and In Vivo: Methods and Protocols
Wash slides 2 × 5 min in PBS. Wash slides in DNase-free water for 5 min. Treat tissue section with Proteinase K solution (see Notes 8 and 9). It will be necessary to calculate the working volume of Proteinase K solution assuming that 50 is required to cover 2 × 1 cm2 tissue area. Dilute Proteinase K concentrate with DNase-free water using the ratio recommended in the TUNEL kit data sheet. Apply Proteinase K solution onto tissue section and cover them gently with the coverslip of appropriate size.
The first technique was initially introduced by Wood et al. (6, 7). It employs non-fluorescent detection and labels apoptosis in unfixed, fresh-frozen sections and in cultured cells. The second protocol was developed by Tanaka and co-authors (3). It uses fluorescence labeling and is applicable to paraffinembedded sections. This technique can also be performed in a double-staining format with immunohistochemistry for additional verification of cell death by apoptosis-related antibodies. The formalin-fixed paraffin-embedded sections generally provide significantly better morphology compared to frozen sections and permit individual morphologic evaluations of labeled cells.
J. Histochem. Cytochem. 41, 7–12. , Gong, J. and Darzynkiewicz, Z. (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. , Marino, S. and Schiffer, D. (1994) A study of apoptosis in normal and pathologic nervous tissue after in situ end-labeling of fragmented DNA. J. Neuropathol. Exp. Neurol. 53, 606–616. , Bursch, W. and Schulte-Hermann, R. (1995) In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis and autolytic cell death: a cautionary note.
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