Akademik Veri Yönetim Sistemi
JOURNAL OF HISTOTECHNOLOGY, cilt.26, sa.3, ss.147-151, 2003 (SCI-Expanded)
The objective of this study was to implement a simple and reliable fluorescent staining method specific to DNA for the visualization of cell nuclei using 488-nm and 543-nm visible lasers or conventional mercury lamps. The staining properties of two different reagents, chromomycin A(3) (C-A(3)) and 7-aminoactinomycin-D (7-AA-D), were analyzed in several types of mammalian cells and tissues with different fixation and preparation methods. Results were obtained using a scanning laser confocal microscope. Experiments were initially conducted in paraformaldehyde-fixed cell cultures to optimize the staining conditions. C-A3 (100 mug/mL) and 7-AA-D (10 muM) showed consistent and specific binding to nuclear DNA and emitted green (similar to580 nm) and red (similar to647nm) colors, respectively. No dose change was needed for formaldehyde-fixed cells in the same conditions. In paraffin-embedded or frozen sections, C-A3 exhibited optimum labeling at 140 mug/mL, whereas 7-AA-D was found optimum at 10 muM concentration. Both dyes displayed similar staining patterns regarding the specificity to chromatin and chromosomes but not to the RNA molecules. Major RNA-containing regions in cells, such as nucleolus or cytoplasm, were entirely devoid of C-A3 and 7-AA-D staining. All stages of mitotic chromosomes, as well as picnotic and apoptotic cells, were successfully labeled. Penetration of dyes for labeling giant cells (e.g., mouse oocytes) was achieved by a mild detergent extraction procedure. C-A3 and 7-AA-D are highly specific, easily handled fluorescent markers in PFA and FA fixed cultured cells, paraffin-embedded, and frozen sections. They can easily be the first choice for a specific DNA labeling as a primary or a counterstain in any fluorescent microscope, especially in confocal microscopes lacking UV laser.