Spurr resin

Freshly dissected chick metatarsal tendons were cut into 3-mm lengths and frozen to −196°C using an EM PACT high pressure freezer (Leica). Freeze substitution for ultrastructure was performed using an AFS system (Leica), starting at –90°C in 2% wt/vol osmium tetroxide in actone, going through pure acetone at −50°C and ending in several changes of Spurr's resin (Spurr, 1969 (link)). at 20°C. Polymerization in fresh resin was then performed at 60°C for 24 h. Freeze substitution for immunolabeling was performed using an AFS system (Leica) using pure acetone at –90°C, pure ethanol at −50°C in ethanol, and ending in several changes of HM20 Lowicryl resin at −50°C. UV polymerization in fresh resin was then performed at –50°C for 48 h and continued at 20°C for 48 h.
Embryonic mouse tails were fixed in 2% glutaraldehyde in 100 mM phosphate buffer, pH 7.0, for 30 min at RT. The tails were then diced and fixed for 2 h at 4°C in fresh fixative. After washing in 200 mM phosphate buffer they were fixed after in 1% glutaraldehyde and 1% OsO₄ in 50 mM phosphate buffer, pH 6.2, for 40 min at 4°C. After a rinse in distilled water they were en bloc stained with 1% aqueous uranyl acetate for 16 h at 4°C, dehydrated and embedded in Spurrs' resin.
Ultra-thin sections for normal transmission electron microscopy were collected on uncoated copper 200 grids, serial sections for 3-D reconstruction on formvar-coated copper 1,000 μm slot grids (stabilized with carbon film) and ultra-thin sections (∼60 nm) for immunolabeling on formvar-coated nickel 400 grids. A postembedding labeling technique was used to detect type I collagen using a rabbit anti–chicken collagen-I antibody (Biodesign International) at a dilution of 1:500 followed by a gold-conjugated goat anti–rabbit antibody (British Biocell International) at a dilution of 1:200. All sections were subsequently stained with uranyl acetate and lead citrate, and examined using either a JEOL 1200EX, Philips EM 400, or Philips BioTwin transmission electron microscope. Images were recorded on 4489 film (Kodak) and scanned using an Imacon Flextight 848 scanner (Precision Camera & Video). Images from EM serial sections were aligned and reconstructed in IMOD for Linux (Kremer et al., 1996 (link)) and visualized using OpenSynu for Linux (Hessler et al., 1992 (link)).

Samples were fixed overnight in a solution of 2.5% glutaraldehyde and 2% PFA in 0.1 M phosphate buffer (pH 7.2-7.4). Samples were washed of fixative 3 times with 0.1 M phosphate buffer for 15 minutes, before being incubated for 1 hour in 1% Osmium Tetroxide, followed by 3 more 15-minute phosphate buffer washes. Samples were then dehydrated via sequential 25-minute washes in 50%, 70%, and 90% ethanol, and 3 washes in 100% ethanol. Spurr Resin was introduced via wash in 1:1 mixture of 100% ethanol and Spurr resin, followed by an overnight wash in pure Spurr resin. Spurr resin was replaced twice the following day before samples were placed in block molds and embedded in fresh Spurr and cured in a 70°C oven overnight. Excess resin was then trimmed away with razor blades to expose embedded tissues for ultrathin sectioning on an Ultracut E ultramicrotome (Reichert-Jung). Sections were placed on a copper grid and treated with uranyl acetate for 20 minutes before being rinsed and treated with lead citrate for 7 minutes and rinsed. Stained sections were imaged using a Phillips FEI Morgagni 268 Transmission Electron Microscope, operating at 80 kV. Images were captured with a Gatan Orius CCD camera.