To evaluate the effect of pH variation on bacterial cell morphology, TEM was carried out as previously described by Schooling and Beveridge (2006) (link) with some modifications. 50 μL of bacterial cell suspension (30 mg/mL) was applied on formvar carbon-coated grids and allowed to dry at room temperature (RT) for 10 min. The coated grid containing the bacterial cells was stained with 2% uranyl acetate for 40 s, followed by washing with distilled water to remove excess stain and drying at RT. Micrographs were recorded using a Tecnai 12BT (FEI) transmission electron microscope at an acceleration voltage of 120 KV. TEM analysis was repeated at least three times for each of the conditions, i.e., growth of cells at pH 6.5 and pH 3.5. A total of 7–9 fields were observed per condition and the total number of MVs per field was determined.
Tecnai 12bt
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tecnai 12BT is a transmission electron microscope (TEM) designed for material science and life science applications. It features a 120 kV electron beam and a bottom-mounted CCD camera for high-resolution imaging. The Tecnai 12BT is capable of providing detailed structural information at the nanometer scale.
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7 protocols using tecnai 12bt
1
Bacterial Cell Morphology and pH
2
Preparation of R3M cells for TEM analysis
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Preparation of R3M cells for TEM experiments were carried out as described by Zuppini et al. (2007) (link). The cell ultrastructure was observed using a Tecnai 12-BT transmission electron microscope (FEI, Eindhoven, the Netherlands) operating at 120 kV equipped with a Tietz camera.
3
Negative Staining of Bacterial Cells
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Negative staining was performed as follows, a 50 µl of planktonic bacterial suspension was air dried onto a glow discharged formvar/carbon coated support grid. Grids were then negatively stained with 2% phosphotungstic acid (pH 6.6) and immediately examined under a FEI Tecnai 12BT transmission electron microscope (FEI, USA) at 80 kV.
4
Immunoelectron Microscopy of TPC1 Protein
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Immunoelectron microscopic analyses were essentially performed as described (Neesen et al., 2002 (link)). Briefly, mouse testes samples of TPC1 wild-type and TPC1-knockout animals were immersion fixed for 2 h in a fixative consisting of 2% paraformaldehyde and 0.05% glutaraldehyde in 0.05 M sodium cacodylate buffer (0.1 M, pH 7.3). Samples were washed with sodium cacodylate buffer, dehydrated in ethanol and propylene oxide as described (Neesen et al., 2002 (link)), and embedded in LR Gold Resin. Ultrathin sections were cut on a Reichert ultramicrotome equipped with a diamond knife and mounted on 100-mesh gold grids. Grids were incubated with the anti-TPC1NK antibody (6.6 μg/ml), diluted in Tris-buffered saline (TBS; 10 mM Tris/HCl, pH 8.0, 150 mM NaCl) for 12 h at 4°C, washed with TBS three times for 15 min and, incubated with a 1:100 dilution of a goat anti-rabbit IgG conjugated to colloidal 10-nm gold particles (BBInternational, Cardiff, United Kingdom). Then sections were washed again with TBS, briefly treated with alkaline lead citrate and uranyl acetate solutions, and examined in an electron microscope (Tecnai12BT; FEI, Hillsboro, OR).
5
Electron Microscopy Analysis of Bacterial Infection in hBMECs
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hBMECs were infected with bacteria for 1 h at MOI 50 as described above and following infection cells were washed with 0.1 M sodium cacodylate buffer, fixed with 2% glutaraldehyde (Sigma) and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (Sigma). Cells were then washed with 0.1 M sodium cacodylate buffer and post fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (Sigma) for 1 h at 4°C. Following subsequent washes with cacodylate buffer and distilled water, cells were then dehydrated in increasing concentrations of ethanol and propylene oxide and finally embedded in DER 332/732 resin (Electron microscopy services) by polymerization at 70°C. Ultrathin sections (70 nm) were cut using a glass knife on a Leica Microtome (Ultracut R) and stained with 1% uranyl acetate and 0.1% lead citrate and viewed using a transmission electron microscope (Tecnai 12BT, FEI) at 120 KV.
6
Transmission Electron Microscopy Analysis of Nanoparticles
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TEM analyses were performed on a FEI Tecnai 12 BT instrument operated at 120 kV. CUR DHA ME was diluted with Milli-Q water at a ratio of 1:50 and mixed by vortexing on cylcomixer. A drop of the ME was placed on a TEM grid covered by a holey carbon film, stained with 2% uranyl acetate for 10 min and blotted with filter paper to form a thin liquid film on the grid. TEM micrographs were recorded using Image Analysis software and CCD camera (Setthacheewakul et al., 2010 (link)).
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Ultrastructural Analysis of Ferroptosis
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