TEM of photoreceptor terminals was performed as described [73 (link)]. Adult fly heads were dissected in 4% PFA and the retinas were removed. The dissected lamina was fixed in a solution with 4% PFA and 2.5% glutaraldehyde for 2 h on ice, followed by fixation in 1% osmium tetroxide for 1.5 h at 4°C. Tissues were then dehydrated in a series of ethanol dilutions at 4°C (10-min wash in 10, 25, 40, 55, 70, 85, 95, and 30-min wash in 100% ethanol for 5 times). Samples were gradually infiltrated with 2 ratios of ethanol and Eponate 12 (Ted Pella), finally going into 3 changes of pure resin. Samples were allowed to infiltrate in pure resin overnight on a rotator and embedded in Eponate 12 resin (Ted Pella). Thin sections (80 nm) were stained with uranyl acetate and lead-citrate (Ted Pella) and examined using a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a Gatan CCD (4k × 3.7k pixels, USA).
Uranyl acetate and lead citrate
Uranyl acetate and lead citrate are commonly used heavy metal stains in transmission electron microscopy (TEM) sample preparation. Uranyl acetate is used to stain cellular components, enhancing contrast for visualization. Lead citrate provides additional staining and contrast enhancement. Both products are widely used in biological and materials science TEM applications.
1 protocol using uranyl acetate and lead citrate
Ultrastructural Analysis of Drosophila Retina
TEM of photoreceptor terminals was performed as described [73 (link)]. Adult fly heads were dissected in 4% PFA and the retinas were removed. The dissected lamina was fixed in a solution with 4% PFA and 2.5% glutaraldehyde for 2 h on ice, followed by fixation in 1% osmium tetroxide for 1.5 h at 4°C. Tissues were then dehydrated in a series of ethanol dilutions at 4°C (10-min wash in 10, 25, 40, 55, 70, 85, 95, and 30-min wash in 100% ethanol for 5 times). Samples were gradually infiltrated with 2 ratios of ethanol and Eponate 12 (Ted Pella), finally going into 3 changes of pure resin. Samples were allowed to infiltrate in pure resin overnight on a rotator and embedded in Eponate 12 resin (Ted Pella). Thin sections (80 nm) were stained with uranyl acetate and lead-citrate (Ted Pella) and examined using a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a Gatan CCD (4k × 3.7k pixels, USA).
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