A METHOD FOR STAINING OF CELL-NUCLEI IN XENOPUS-LAEVIS EMBRYOS WITH CYANINE DYES FOR WHOLE-MOUNT CONFOCAL LASER-SCANNING MICROSCOPY

To study the cell cleavage pattern in experimentally treated Xenopus l aevis blastulae, we devised a method to visualize all cell nuclei, whe ther in interphase or in a mitotic phase, in whole-mount embryos using confocal laser scanning microscopy. Optimal staining conditions were defined for the recently commercialized cyanine nucleic acid stain TO- PRO-3, which is excited by a 647-nm laser beam and fluoresces in the f ar red of the spectrum. This is beyond the spectral range of autofluor escence caused by most biomolecules and, in particular, by the high am ount of yolk granules in these embryos. The quality of the TO-PRO-3 im age was compared to chat after nuclear staining with BOBO-3, another c yanine dye that fluoresces at slightly shorter wavelengths. In the pro posed procedure, special attention is paid to permeabilization of the membranes to the dyes and to bleaching of the natural pigment of the e mbryos with maximal preservation of cellular and nuclear structures. B ecause of its emission maximum at 661 nm, TO-PRO-3 is a promising nucl ear stain for specimens with special background problems and for multi color fluorescence microscopy.