Reichert ultracut e

Samples were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 (EMS, Hatfield, PA, United States), for at least 1–2 h at room temperature with agitation. Samples were rinsed [3×; 10 min, room temperature (RT)] in 0.1 M sodium cacodylate buffer, pH 7.2, and immersed in 1% osmium tetroxide with 1.6% potassium ferricyanide in 0.1 M sodium cacodylate buffer for 2 h. Samples were rinsed (3×; 10 min, RT) in buffer and then briefly in distilled water (1×; 1 min, RT). Samples were then subjected to an ascending acetone gradient (10 min; 35%, 50%, 70%, 80%, and 90%) followed by pure acetone (2×; 10 min, RT). Samples were progressively infiltrated with Epon resin (EMS, Hatfield, PA, United States), while rocking, and then polymerized at 60°C for 24–48 h in silicon molds. Thick sections (200 nm) were cut using a Reichert Ultracut E (Leica, Wetzlar, Germany), collected on glass slides, stained with toluidine blue, and used for general orientation. Thin sections (70 nm) were collected onto formvar-coated 50-mesh copper grids or slot grids. The grids were post-stained with 2% uranyl acetate followed by Reynold’s lead citrate, for 5 min each. The sections were imaged using a Tecnai 12 120-kV TEM (FEI, Hillsboro, OR, United States) and data were recorded using an UltraScan 1000 with Digital Micrograph 3 software (Gatan Inc., Pleasanton, CA, United States).

Cells were pelleted by centrifugation for 5 min, at 3000 g and 4 °C. After washing with Sorensen’s phosphate buffer (0.1 M, pH 7.4), the pellets were re-suspended with a solution of 4% [v/v] paraformaldehyde and 1.5% [v/v] glutaraldehyde in Sorensen’s phosphate buffer. After fixation for 45 min at room temperature, samples were washed four times with Sorensen’s phosphate buffer at 4 °C and embedded in low-melting-point agarose (1.5–2%). The agarose pellet was solidified at 4 °C and cut into small cubes of about 1 mm edge length. Samples were post-fixed with 1% osmium tetroxide in Sorensen’s phosphate buffer in the dark for 1 h at room temperature. Samples were dehydrated with graded ethanol and propylene oxide and embedded in EMBed-812 resin. Ultrathin sections were cut with an ultramicrotome (Reichert Ultracut E, Leica, Rueil-Malmaison, France) and transferred to copper/palladium grids. The grids were contrasted with uranyl acetate and lead citrate and observed on a Hitachi H7500 TEM (Hitachi Scientific Instruments Co., Tokyo, Japan) operating at 80 kV and equipped with an AMT camera driven by AMT software (AMT, Danvers, USA).

Zebrafish larvae were fixed in 4% paraformaldehyde, 2.5% glutaraldehyde, 0.02% picric acid, 0.1 M Na cacodylate buffer, washed and fixed in 1% osmium tetroxide in 0.1 M Na cacodylate buffer. They were subsequently treated with 0.5% tannic acid followed by 1% sodium sulfate. The pellets were treated with propylene oxide and embedded in Epon/Araldite. Thin sections (70 nm) of the pelleted samples were cut on a Reichert Ultracut E (Leica, Deerfield, IL) using a diamond knife (Diatome, Electron Microscopy Sciences, Hatfield, PA), mounted on parlodion-coated copper slot grids and stained in uranyl acetate and lead citrate. Sections were examined on a Philips CM100 transmission electron microscope (FEI, Hillsbrough, OR). Images were documented and measurements were taken using a Megaview III CCD camera (Olympus Soft Imaging Solutions, Lakewood CO). Transverse sections were obtained through the trunk muscle region, the yolk and the eye region.