Gagna Claude E, Kuo Hon-Reen, Chan Norman J, Mitacek Eugene J, Spivak Alla, Pasquariello Tiffany D, Balgobin Chandrika, Mukhi Ruhayna, Lambert W Clark
New York Institute of Technology, Department of Life Sciences, New York College of Osteopathic Medicine, Old Westbury, NY 11568-8000, USA.
J Histochem Cytochem. 2007 Oct;55(10):999-1014. doi: 10.1369/jhc.7A7194.2007. Epub 2007 May 28.
Histotechnological processing of DNA can cause damage to and loss of DNA and can change its structure. DNA probes have severe tissue-staining limitations. New DNA probes and improved histotechnology are needed to enhance the characterization of fixed tissue-bound DNA. Our team developed a novel DNA staining technique and histotechnological processing procedure that improves tissue-bound DNA retention and the qualification and quantification of intact double-stranded (ds)-B-DNA. We used the ultrasensitive PicoGreen ds-DNA probe for the histochemical characterization of ds-DNA. Fifteen fixatives were examined to determine which were best for preventing DNA denaturation and retaining original DNA content and structures. Our use of a microwave-vacuum oven reduced heating temperatures, shortened heating and processing times, and enhanced fixation. We achieved better qualitative and quantitative results by using superior tissue-acquisition techniques (e.g., reduced prefixation times) and improved histotechnology. We also compared our novel approach with archival tissues, delayed fixation, less sophisticated and conventional histological processing techniques, and by experimenting with preservation of tissue-bound ds-Z-DNA. Results demonstrate that our histotechnological procedure and nucleic acid staining method significantly improve the retention of intact, undamaged ds-DNA which, in turn, allows the investigator to more precisely quantify the content and structures of unaltered and undamaged tissue-bound ds-B-DNA.
DNA的组织技术处理可能会导致DNA的损伤和丢失，并可能改变其结构。DNA探针存在严重的组织染色局限性。需要新的DNA探针和改进的组织技术来加强对固定组织中结合DNA的表征。我们的团队开发了一种新型的DNA染色技术和组织技术处理程序，可改善组织结合DNA的保留以及完整双链（ds）-B-DNA的鉴定和定量。我们使用超灵敏的PicoGreen双链DNA探针进行双链DNA的组织化学表征。检测了15种固定剂，以确定哪种最适合防止DNA变性并保留原始DNA含量和结构。我们使用微波真空烤箱降低了加热温度，缩短了加热和处理时间，并增强了固定效果。通过使用优质的组织获取技术（例如，减少预固定时间）和改进的组织技术，我们获得了更好的定性和定量结果。我们还将我们的新方法与存档组织、延迟固定、不太复杂的传统组织学处理技术进行了比较，并对组织结合的ds-Z-DNA的保存进行了实验。结果表明，我们的组织技术程序和核酸染色方法显著提高了完整无损的双链DNA的保留率，这反过来又使研究人员能够更精确地量化未改变和未受损的组织结合双链B-DNA的含量和结构。
