Cube u mwu

Modified double staining with fluorescein diacetate (FDA) and propidium iodide (PI) was performed to determine the viability of the ESEs [17 (link)]. FDA causes green fluorescence in viable cells because the non-fluorescent FDA easily penetrates into viable cells and is hydrolysed to a brightly fluorescent fluorescein (λ_excitation = 490 nm and λ_emission = 514 nm) that does not readily diffuse through the cytoplasmic membrane. The red fluorescence of PI (λ_excitation = 536 nm and λ_emission = 620 nm) in cells shows that these cells are dead because this compound cannot pass through the functional cytoplasmic membrane. In our experiments, ESEs (~1.0 mg) were harvested and diluted with water to a final volume of 50 μl. The stock solutions of PI and FDA were added to a final concentration of 20 μg/ml and 1.0 μg/ml, respectively. After 5 min of incubation at room temperature, the percentages of dead (red-stained cells) and viable cells (green-stained cells) were evaluated using an Olympus AX 70 fluorescence microscope with an Olympus cube U MWU coupled with a digital camera. The percent quantification of red (dead cells) and green areas (viable cells) in the compact embryonic group of single embryos was determined from the acquired digital images using an image analysis method (Image–Pro Plus, ver. 1.3, Sony).

For observation of fluorescence of GFP, transformed cells were collected by centrifugation at 200×g for 10 min, and examined by a fluorescence microscope BX-60 (Olympus, Tokyo, Japan), using the cube U-MNIBA (Olympus). When GFP fluorescence was faint, we performed immunofluorescence detection using anti-GFP antibody, essentially according to Nishida et al. (2004) (link). In the reaction with antibody, an anti-GFP monoclonal antibody was diluted to 1/200 with immunoreaction enhancer solution (Can Get Signal immunostain solution B, TOYOBO, Osaka, Japan) and reacted for 1 h. As the secondary antibody, anti-mouse monoclonal antibody labelled with Alexa fluor 488 (Invitrogen, Carlsbad, CA) was diluted to 1/200 with immunoreaction enhancer solution, and reacted for 30 min. The immunostained cells were observed by the fluorescence microscope with the cube U-MNIBA (Olympus) for Alexa fluor 488, the cube U-MWIG (Olympus) for observation of autofluorescence of chlorophyll, and the cube U-MWU (Olympus) for observation of DAPI-stained DNA. Nomarski differential interference image was also recorded. All microscopic images were captured by a digital camera (model DP-70, Olympus).