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 laevis blastulae, we devised a method to visualize all cell nuclei, whether 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 far red of the spectrum. This is beyond the spectral range of autofluorescence caused by most biomolecules and, in particular, by the high amount of yolk granules in these embryos. The quality of the TO-PRO-3 image was compared to that after nuclear staining with BOBO-3, another cyanine dye that fluoresces at slightly shorter wavelengths. In the proposed procedure, special attention is paid to permeabilization of the membranes to the dyes and to bleaching of the natural pigment of the embryos with maximal preservation of cellular and nuclear structures. Because of its emission maximum at 661 nm, TO-PRO-3 is a promising nuclear stain for specimens with special background problems and for multicolor fluorescence microscopy.
de Mazière
A M
AM
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands.
Hage
W J
WJ
Ubbels
G A
GA
D000818:Animals; D002232:Carbocyanines; D002463:Cell Membrane Permeability; D002467:Cell Nucleus; D004396:Coloring Agents; D004625:Embryo, Nonmammalian / Q000166:cytology; D005456:Fluorescent Dyes; D018613:Microscopy, Confocal / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation; D014982:Xenopus laevis / Q000196:embryology
C098829:BOBO 3; D002232:Carbocyanines; D004396:Coloring Agents; D005456:Fluorescent Dyes; C098830:TO-PRO-3