Fei tecnai g2 20 twin 200 kv lab6 tem

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).

Samples were stained with 1% Toluidine Blue (Fishersci.com) before being mounted on microslides (Gold‐Seal, Fishersci.com). The number of single cells and clusters of 2–4, 5–10 and >10 cells were counted from micrographs obtained using an Olympus BX43 microscope with an Olympus DP26 camera. Proportions of each cluster were determined from greater than 1,000 cells from 20 micrographs from 3 replicate experiments for each genotype and treatment.
For electron microscopy, untreated and treated samples were infiltrated with Embed‐812 epoxy resin (Electron Microscopy Sciences, Hatfield, PA) for 24 h, spun down into a pellet in a 2‐mL vial and polymerized at 70 °C overnight. Semi‐thin Toluidine Blue‐stained sections were mounted on glass slides for light microscopy (500 nm thickness), then ultrathin sections (200 nm and 100 nm) were collected on 100‐mesh formvar‐coated copper grids, stained with 4% aqueous uranyl acetate, rinsed in water, and finally stained with lead citrate and rinsed. Images were captured with a four megapixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on an FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hillsboro, OR).

Particles comprising vascular tissue were visually selected from milled native or milled and DA/5 mM Fe³⁺ pretreated corn stover samples under the anaerobic setup condition. The particles were processed via the microwave resin embedding process described previously [23 ]. Briefly, samples were first infiltrated with water, followed by dehydration with increasing concentrations of ethanol with intermittent microwave steps. Next, the samples were infiltrated with LR White (London Resin Company, Reading, United Kingdom) by incubating at room temperature for several hours (up to overnight) in increasing concentrations of resin diluted in ethanol. Microwave steps were performed each time the concentration was increased. The fully infiltrated samples were capsules and placed in a laboratory oven at 60 °C overnight to polymerize the resin. Ultra-thin (~60 nm) sections were cut using a Leica Ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and collected on 0.5% polyvinyl formvar coated copper slot grids (SPI Supplies, West Chester, PA, USA). Grids were individually stained for 30 s with 1% aqueous KMnO₄. Images were taken with a four-megapixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA, USA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hillsboro, OR, USA) operated with an accelerating voltage of 200 kV.