Carboxyfluorescein as a Marker at Both Light and Electron Microscope Levels to Follow Cell Lineage in the Embryo

Abstract

Lipid-soluble dyes have been found to be useful markers for studying cell lineages in embryos (1,2). They have the advantage of diffusing into or across the plasmalemma, eliminating the need for microinjection. DiI (1,1 dioctadecyl 3,3,3′,3′ tetramethylindocarbocyanine perchlorate) is irreversibly incorporated into the plasmalemma and has been used extensively to follow cell fates, but it has the disadvantage of not surviving fixation or wax embedding (2–4). The frozen sections of unfixed tissue that must be examined lack cytological detail. On the other hand, 5, 6 carboxy 2′,7′ dichloro-fluorescein diacetate succinimidyl ester (CCSFE), a carboxyfluorescein derivative that resists bleaching (5) better than the monochloro-form (1), is a stable cytoplasmic marker that stands up to formaldehyde fixation and wax embedding. In this report, we show that CCSFE also tolerates glutaraldehyde and osmium tetroxide fixation, embedding in epoxy resin, sectioning, and transmission electron microscopy (TEM).