Formvar coated slot grids

Pretreated biomass samples were dehydrated by treating with increasing concentrations of acetone by increments of ~20 vol % and intermittently heated for 1 min in a Pelco microwave oven after each addition of acetone. After dehydration, the samples were infiltrated with Eponate 812 (EMS, Hatfield, PA) by incubating at room temperature for several hours to overnight in increasing concentrations of resin in increments ~20 vol % diluted in acetone until 100 % resin was reached, after which three complete resin exchanges were performed. The infiltrated samples were transferred to capsules and the resin polymerized in and oven at 60 °C overnight. Samples embedded in resin blocks were sectioned to ~250 nm with a Diatome diamond knife on a Leica EM UTC ultramicrotome (Leica, Wetzlar, Germany). Sections were collected on 0.5 % Formvar coated slot grids (SPI Supplies, West Chester, PA) and were post-stained for 2 min with 1 % aqueous KMnO4. Images were captured with a four mega-pixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hillsboro, OR) using SerialEM [40 (link)].

Samples of Arabidopsis stem were prepared for transmission electron microscopy (TEM) by high-pressure freezing and freeze substitution according to the literature (Donohoe et al., 2006 (link)). Briefly, samples were cryo-preserved using high-pressure freezing in a Leica EM-Pact2 and freeze substituted in 1% OsO₄ over several days in a Leica AFS2 (Leica, Wetzlar, Germany). Samples were infiltrated with Eponate 812 (EMS, Hatfield, PA) by incubating at room temperature for several h to overnight in increasing concentrations of resin (15, 30, 60, 90%, 3 × 100% resin, diluted in acetone). The samples were transferred to capsules and the resin polymerized in an oven at 60°C overnight. Resin embedded samples were sectioned to approximately 50 nm with a Diatome diamond knife on a Leica EM UTC ultramicrotome (Leica, Wetzlar, Germany). Sections were collected on 0.5% Formvar coated slot grids (SPI Supplies, West Chester, PA). Grids were post-stained for 4 min with 2% aqueous uranyl acetate and for 2 min with Reynold's lead citrate. Images were taken with a 4 mega-pixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hillsboro, OR).

LR White embedded samples were sectioned to ~60 nm with a Diatome diamond knife on a Leica EM UTC ultramicrotome (Leica, Wetzlar, Germany). Sections were collected on 0.5 % (v/v) Formvar-coated slot grids (SPI Supplies, West Chester, PA). All grids were post-stained for 4 min. with 2 % (w/v) aqueous uranyl acetate and 2 min. with Reynolds lead citrate. Images were taken with a 4 mega-pixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hilsboro, OR).