Flash 1400 microscope

Zebrafish embryos at 10 somites stage (~14 hpf) were fixed with a standard TEM fixative solution of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M HEPES at 4°C overnight. Samples were washed with HEPES buffer and post fixed in 1% osmium tetroxide in 0.1 M HEPES for 2 hr. After post fixation, samples were rinsed with 0.1 M HEPES three times and treated with 1% tannic acid in 0.1 M HEPES buffer for 1 hr. Consecutively, samples were washed thoroughly with water three times. Samples were treated with series of ethanol solutions (50%, 75% ethanol) for ten minutes. Embryos were processed for enblock staining with 1% uranyl acetate in 75% ethanol for 1 hr on ice. After enblock staining, samples were processed for dehydration in a series of ethanol solutions (85%, 90%, 95%) on ice. The final dehydration procedure was done at room temperature using 100% ethanol twice for 15 minutes. Dehydration was continued with propylene oxide for 15 minutes two times. Subsequently, infiltration was done using propylene oxide and epon resin mixture. Later 100% epon was used for overnight infiltration. Embryos were changed into 100% fresh epon resin two times before embedding. Samples were embedded in 100% resin and polymerized at 64°C. Ultrathin sections, with thickness of 60 nm, were collected, stained with lead citrate solution, and imaged with a JEOL Flash-1400 microscope.