Morgagni 268 transmission electron microscope

UROtsa cells and bladder specimens were rinsed with 0.2M Cacodylate pH 7.4 for 5 min and fixed with 2% glutaraldehyde in 0.1M Cacodylate. Specimens were post fixed using 1% osmium tetroxide in 0.1M cacodylate for 1 h at 4°C. Fixed specimens were given a wash with 7% sucrose overnight and dehydrated for 15 min at graded concentrations (50, 70, 90 & 100%) of ethanol and then incubated in propylene oxide for 10 min followed by infiltration of specimen with 1:1 (propylene oxide:812 epoxy resin) for 1 h. Subsequent steps involved embedding in block and polymerization at 60°C overnight. Ultra-thin sections were cut with Ultra-microtome RMC MT-7000, Tucson, AZ, which was negatively stained with uranyl acetate with silver enhancer. Images were acquired using Morgagni 268 Transmission Electron Microscope FEI Company, Hillsboro, OR.

Cells were cultured in permanox petri dish and treated with gemcitabine (1 μmol/L) and C-DIM12 (15 μmol/L) for 24 hours. The cells were fixed with 2.5% paraformaldehyde, 2% glutaraldehyde, 0.1 mol/L cacodylate buffer, and embedded using Epon 812. The ultra-thin sections (∼100 nm) were cut using a Leica EM UC6 ultramicrotome and diamond knife. The sections were then placed on copper grids, poststained with saturated Uranyl Acetate and Reynolds Lead Citrate, and imaged using an FEI Morgagni 268 transmission electron microscope equipped with a MegaView III CCD camera.

Transmission electron microscopy (TEM) was performed on small (1 mm³) lung tissue samples, which were processed according to a routine Epon-embedding procedure as previously described [20 (link),24 (link)–26 (link)]. Briefly, mouse lung samples were fixed by immersion in 4% glutaraldehyde, post-fixed in 1% OsO₄ and further processed for Epon embedding. Thin sections (60 nm) were double-stained with uranyl acetate and lead citrate and examined under a Morgagni 268 transmission electron microscope (FEI Company, Eindhoven, The Netherlands) at 80 kV. Digital electron micrographs were recorded with a MegaView III CCD using iTEM-SIS software (Olympus, Soft Imaging System GmbH, Münster, Germany).

As western blot analysis for direct confirmation of Cry1Ba1 protein expression was confounded by the presence of high levels of phenolics in B. koenigii, an indirect approach to confirm pesticidal protein expression was adopted. Transmission electron microscopy was used to examine the guts of Asian citrus psyllids (ACP), Diaphorina citri, fed on transgenic B. koenigii, or WT plants, to look for gut damage associated with exposure to Cry1Ba1. A total of 20 adult ACP per plant were maintained on the plants for 8 days. A total of ≥40 live ACP adults were then collected and pooled per treatment for TEM analysis. The psyllids were dissected to remove the head and the tip of the abdomen, and then fixed for 16 h at 4°C in 1X PBS buffer containing 3% (v/v) glutaraldehyde, postfixed for 4 h at room temperature in 2% (v/v) osmium tetroxide, dehydrated in acetone, and embedded in Spurr’s Resin (Spurr, 1969 (link)). Light microscopy slides were prepared for orientation purposes to locate the midgut in cross section starting from the anterior or posterior ends of 8–10 psyllids from the WT and from each transformed plant. For TEM, 90 nm ultrathin sections were mounted on copper grids, stained with 2% (w/v) aqueous uranyl acetate and lead citrate (Reynolds, 1963 (link)), and observed using an FEI Morgagni 268 Transmission Electron Microscope (FEI Company, Hillsboro, Oregon, United States).

Two to 3 mm squared pieces of the above samples were prepared for TEM by placing them in 3% glutaraldehyde fixative and further processing them in the laboratory. The samples were rinsed in buffer, post-fixed in osmium as above, and rinsed again in buffer before dehydration in acetone (10% steps, 10 min for each step). Subsequently the samples were infiltrated in Spurr’s resin (Spurr 1969 (link)) over a 3 day period, placed in molds and hardened in an oven at 70 °C. One micrometer thick sections were prepared with a glass knife on a LKB Huxley Ultramicrotome (LKB Instruments, Sweden), stained with methylene blue/azure A and counter stained with basic fuchsin (Humphrey and Pittman 1974 (link)) for light microscopy. Sections were observed under an Olympus BX61 compound microscope (Cambridge Scientific Products, Watertown, MA, USA) and photographed using an OMAX CMOS 14mp digital camera. Ultrathin sections for TEM were prepared with a diamond knife on the same ultramicrotome, stained with 2% aqueous uranyl acetate, post-stained with Reynolds lead citrate (Reynolds 1963 (link)), and photographed using a Morgagni 268 transmission electron microscope (FEI Company, The Netherlands).

Small pieces of explanted heart tissues were immersion-fixed in 150 mM HEPES (pH 7.35) containing 1.5% formaldehyde and 1.5% glutaraldehyde (GA). Tissue embedding and heart section preparation were done at the Institute of Functional and Applied Anatomy, Hannover Medical School (MHH) as previously described.⁴⁷ (link) The Phoenix hiPSC-CM inserts were fixed in fixative solution composed of 1.5% PFA, 1.5% GA, and 0.15 M HEPES (pH 7.35) and processed further as described previously.⁴⁸ (link) Several grids were prepared from different regions of the insert. The entire grid (from three different regions of one insert/condition) was imaged to sample in an unbiased approach. Electron microscopic examinations were performed at the Hannover Medical School, electron microscopy facility, with the FEI Morgagni 268 transmission electron microscope (FEI, Eindhoven, the Netherlands) operated at 80 kV using a Veleta CCD camera (Olympus Soft Imaging Solutions).