Novel DNA staining method and processing technique for the quantification of undamaged double-stranded DNA in epidermal tissue sections by PicoGreen probe staining and microspectrophotometry.
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.
Gagna
Claude E
CE
New York Institute of Technology, Department of Life Sciences, New York College of Osteopathic Medicine, Old Westbury, NY 11568-8000, USA. dr.c.gagna@att.net
Kuo
Hon-Reen
HR
Chan
Norman J
NJ
Mitacek
Eugene J
EJ
Spivak
Alla
A
Pasquariello
Tiffany D
TD
Balgobin
Chandrika
C
Mukhi
Ruhayna
R
Lambert
W Clark
WC
D000818:Animals; D004247:DNA / Q000032:analysis*; D004817:Epidermis / Q000737:chemistry*; D005404:Fixatives; D005456:Fluorescent Dyes; D016591:Histocytological Preparation Techniques / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D016612:Paraffin Embedding; D011003:Ploidies; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013552:Swine
D005404:Fixatives; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA