Glutaraldehyde

SEM analysis of the biomaterials was performed prior to differentiation and after DPPSC and SAOS-2 cells were differentiated on the biomaterials. The samples were fixed with 2.5% glutaraldehyde (Ted Pella Inc.) in 0.1 M Na-cacodylate buffer (EMS, Electron Microscopy Sciences, Hatfield, PA, USA) (pH 7.2) for 1 h on ice. After fixation, the samples were treated with 1% osmium tetroxide (OsO₄) for 1 h. The samples were then dehydrated in serial solutions of acetone (30–100%) with the scaffolds mounted on aluminum stubs. The samples were examined with a Zeiss 940 DSM scanning electron microscope.

The cells were fixed for 30 min with 2.5% glutaraldehyde (Ted Pella Inc., Redding, CA, USA), washed with PBS (pH 7.2–7.4), and postfixed with 1% OsO4 solution in PBS (pH 7.2–7.4; Sigma-Aldrich) for 1 h. Dehydration and embedding in Epon 812 (Sigma-Aldrich) were performed using the standard technique. Ultrathin sections of Epon-embedded samples were stained with 1.5% uranyl acetate solution and lead citrate according to Reynolds [37 (link)] and analyzed with a JEM-1011 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a GATAN ES500W digital camera operated by the Digital Micrograph GATAN software.

For transmission electron microscopy (TEM), samples were fixed quickly for 24 hours in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, Missouri, USA), 2.5% glutaraldehyde (Ted Pella, Redding, California, USA), and 0.003% CaCl2 (Sigma-Aldrich) in sodium cacodylate buffer (Sigma-Aldrich), post-fixed in 2% osmium tetroxide (Ted Pella), dehydrated in an acetone dilution series (30%, 50%, 70%, and 90%, followed by 3 × 100%), and embedded with Eponate 12 resin (Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). Sections for TEM were cut to a thickness of 70 nm with diamond knives and a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany). The sections were stained with either 2% (w/v) aqueous uranyl acetate and 1% (w/v) lead citrate. Specimens were viewed with a TecnaiG2 Spirit 120kV (Thermo Fisher, Waltham, Massachusetts, USA) transmission electron microscope.
Due to size variability between wild-type and eas-1(lf) animals (S1B Fig), the relative position of the AMsh cell body on the animal was used to determine where to section the animals. The relative position of the AMsh cell body was measured to be consistent and similar between both genotypes, at around 0.12 times the total length of the animal from the nose tip (S1J Fig). Sections around this region were used for TEM imaging, and the AMsh cell bodies were identified in reference to micrographs from WormAtlas [76 ].

The optic nerve heads of three rats from each group were separated on ice and immediately fixed in 2.5% glutaraldehyde (Ted Pella, Redding, CA, United States) in 0.1 M phosphate buffer at 4°C. After rinsing in 0.1 M phosphate buffer for three times, tissues were post-fixed in 1% osmic acid at 4°C for 2 h, washed for three times, dehydrated in ascending grades of ethyl alcohol, and embedded in epoxy resin. Then sections on unmyelinated optic nerve were sliced and then viewed using a transmission electron microscope.

After fixation of arterial samples in 2.5% glutaraldehyde (TED PELLA, CA, USA) in PBS (pH 7.2), specimens were post-fixed in 1% osmium tetroxide (Heraeus, Hanau, Germany), dehydrated in graded ethanol and propylene oxide (Acros Organics, USA), and then embedded in Epoxy resin (mix with Nadic Methyl Anhydride (NMA) and Dodecenyl Succinic Anhydride (DDSA) and DMP-30, all reagents from Polysciences (PA, USA). Serial ultrathin sections were cut using an LKB-III ultratome (LEICA, Wetzlar, Germany). Ultrathin sections were stained with uranyl acetate (TED PELLA, CA, USA) and lead citrate (TED PELLA, CA, USA) and were examined with the aid of a Hitachi H7600 electron microscope (Hitachi, Japan) at an accelerating voltage of 100 kV.

Roswell Park Memorial Institute (RPMI) 1640 culture medium, phosphate saline buffer (PBS), 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) (0.5 g L⁻¹, in PBS), and menadione (for 3 μM final concentration) (Pierce et al., 2008 (link)) were acquired from Sigma-Aldrich (MO). Osmium tetroxide (OsO₄; 4% solution) and glutaraldehyde (2.5% solution) were acquired from Ted Pella, Inc. Solutions of the different reagents were prepared in Milli-Q water.