Distilled em grade

Ciona ovaries were fixed in 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) (Distilled EM grade, Electron Microscopy Sciences, Hatfield, PA, USA) in 0.1M phosphate buffer (PB) pH 7.4 at 4 °C for 1h. After washing, the samples were dehydrated and infiltrated with a 50:50 mixture of ethanol and resin, transferred to a fresh 100% resin (LR white, London Resin, Berkshire, UK), and polymerized by an ultraviolet polymerizer. Ultra-thin sections of the polymerized resins were mounted on nickel grids and incubated with anti-PEP51 antibody, followed by 15nm gold particle-labelled secondary antibody. The grids were placed in 2% GA in 0.1 M PB and dried, then stained with 2% uranyl acetate for 15 min and a Lead stain solution (Sigma-Aldrich, Tokyo, Japan). The samples were observed under a transmission electron microscope (JEM-1400Plus, JEOL, Tokyo, Japan) at 100 kV. Digital images were obtained with a CCD camera (EM-14830RUBY2, JEOL). Immunoelectron microscopy was carried out by Tokai Electron Microscopy, Inc. (Nagoya, Japan).

The samples were fixed with 2% paraformaldehyde (PFA), 2% glutaraldehyde (Distilled EM grade, Electron Microscopy Sciences, Hatfield, PA) in 0.1 M PBS pH 7.4 at 4°C overnight. After the fixation, the samples were rinsed three times with 0.1 M PBS for 30 minutes, followed by post-fixation with 2% osmium tetroxide(OsO₄) in PBS at 4°C for two hours. The samples were then infiltrated with propylene oxide (PO) twice for 15 min and put them into a mixture of PO and resin (Quetol-812; Nisshin EM Co.,Tokyo, Japan) for one hour, followed by keeping the cap of tube open, and PO was volatilized overnight. The samples was transferred to a fresh 100% resin, and polymerized at 60°C for 48 hours. The blocks were ultra-thin sectioned at 70 nm with a diamond knife using a ultramicrotome (ULTRACUT UCT; Leica, Wetzlar, Germany) and sections were placed on copper grids. They were stained with 2% uranyl acetate at room temparature for 15 min. and then rinsed with distilled water followed by being secondary-stained with Lead stain solution (Sigma-Aldrich) at room temparature for three minutes. The grids were observed by a transmission electron microscope (JEM-1200EM; JEOL Ltd., Akishima, Japan) at an acceleration voltage of 80 kv. Digital images (2048×2048pixels) were taken with a CCD camera (VELETA; Olympus Soft Imaging Solutions GmbH, Münster, Germany).

For scanning electron microscopy (SEM), high-resolution images were obtained using a scanning electron microscope (SEM JCM-5000; JEOL Ltd., Tokyo, Japan) operating at 10 kV. A 5 nm thick platinum layer was deposited on the samples by sputtering (MSP 30T; Showa Shinku Device, Sagamihara, Japan). The angular distributions and fiber diameters were evaluated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
For transmission electron microscopy (TEM), the samples were fixed with 2% glutaraldehyde (Distilled EM Grade, Electron Microscopy Sciences, Hatfield, PA, USA) in NaHCa buffer (100 mM NaCl, 30 mM HEPES, 2 mM CaCl₂, adjusted to pH 7.4 with NaOH) and then postfixed with 0.25% osmium/0.25% K₄Fe(CN)₆, with 1% tannic acid and finally with 50 mM uranyl acetate. The samples were then washed, dehydrated in a series of ethanol solutions, and embedded in TABA EPON 812 resin (TAAB Laboratories Equipment Ltd., Reading, UK). After polymerization at 65°C, ultra-thin sections (60–100 nm) were cut vertical to the PMGI fiber orientation using an ultramicrotome (Leica FC6, Vienna, Austria). The sections were then mounted on EM grids, stained with lead citrate, and observed by TEM (JEOL JEM1400).