Epoxy tissue stain

Dissected brachial plexus was placed in 4% PFA overnight then rinsed and transferred to 10 mL pH 7.2 containing 2.5% glutaraldehyde-0.1 M imidazole buffer. Tissues were then immersed in 2 mL 2% osmium tetroxide in imidazole buffer for 4 hr and washed in distilled water followed by dehydration in a graded series of ethanol (50%, 80%, absolute). Sequentially, tissue sections were immersed in a mixture of 1:1 absolute ethanol and propylene oxide and then in propylene oxide in sealed vials on a rotating mixer for 15 min. Finally, tissue was infiltrated with a 1:1 mixture of propylene oxide and epoxy resin overnight in open vials then embedded with epoxy resin in molds at 60°C for 48 hr. Embedded tissue was first ‘thick’ sectioned at 0.25 micrometers with glass knives and stained with epoxy tissue stain (Electron Microscopy Sciences, Hatfield, PA). Selected areas were then thin sectioned at 70 nm with diamond knives and mounted on Formvar-Carbon coated grids and stained with 2% uranyl acetate and lead citrate solution. Digital images were collected with a mid-mount XR611 camera (AMT, Inc., Woburn, MA) in a model H-7650 electron microscope (Hitachi High-Technologies, Dallas, TX).

TMJ condyles were excised and fixed for several days in 1.5% glutaraldehyde/1.5% formaldehyde, rinsed, then decalcified in 0.2 M EDTA with 50 mM TRIS in a laboratory microwave (Ted Pella, Inc) operated at 97.5 watts for fifteen, 99 min cycles. Samples were fixed again in 1.5% glutaraldehyde/1.5% formaldehyde with 0.05% tannic acid overnight, then rinsed and post-fixed overnight in 1% OsO4. Samples were dehydrated and extensively infiltrated in Spurr’s epoxy and polymerized at 70 °C^{49 (link)}. One-micron thick sections stained with an epoxy tissue stain (Electron Microscopy Sciences) were used to identify regions of interest. Ultrathin sections containing ROI were cut at 80 nm, contrasted with uranyl acetate and lead citrate, and imaged using a FEI G20 TEM operated at 120 kV with montages collected using an AMT XR-41 2 × 2K camera. The acquired images were stitched using ImageJ software^{50 (link)}.