BODIPY TR methyl ester serves as a lipophilic GFP counterstain, easily permeating cell membranes to accumulate in endomembranous organelles, while exhibiting minimal localization in plasma membranes. It is a highly effective red fluorescent vital dye (Ex=568 nm, Em=625 nm) that can reveal the location and shape of cell nuclei, the morphology of cells in embryonic tissues, and the boundaries of organ-forming tissues within an entire embryo [1].

BODIPY TR methyl ester dye: Solubilized into anhydrous DMSO as a 5 mM stock solution. The stock DMSO solution is diluted 1:50 into Embryo Rearing Medium (ERM) buffered with 5 mM HEPES (pH 7.2), creating a final labeling solution of 100 μM BODIPY TR methyl ester dye with 2% DMSO, stored at -20°C. Embryos are stained in 100 μM BODIPY TR methyl ester dye for 1 h, followed by three successive washes in HEPES-buffered ERM. Embryos are then mounted in an open-faced chamber for imaging. For time-lapse recordings, vitally stained embryos are deyolked and secured to a coverslip. Embryos used for fixation experiments are fixed in 4% paraformaldehyde in HEPES-buffered ERM at 4°C for 1 h, then washed in ERM and mounted for confocal imaging. Samples are analyzed on a confocal microscope (with a 640 × 480 pixel frame buffer and an air-cooled Ar-Kr laser), co-exciting GFP and BODIPY TR methyl ester dye using two wavelengths of the major Ar-Kr laser lines (488 and 568 nm) simultaneously.