Column fractions or particles immunoprecipitated with anti-RNA Pol II CTD4H8 antibodies (EMD Millipore, catalog no. 05-623) were spotted on Formvar Film 150 Mesh Copper grids (Electron Microscopy Sciences, catalog no. FF150-CU) and stained with 1% uranyl acetate (Electron Microscopy Sciences, catalog no. 22400-1). Negative control IP experiments were performed in cell lysates with 15 mg of Normal Mouse IgG (Millipore, catalog no. 12-371). Images were acquired on an FEI Tecnai Spirit 120S transmission electron microscope.
Tecnai spirit 120
Manufactured by Thermo Fisher Scientific
The Tecnai Spirit 120 is a transmission electron microscope designed for high-resolution imaging of a variety of samples. It operates at an accelerating voltage of 120 kV and features a LaB6 electron source, providing enhanced brightness and resolution compared to tungsten filament sources. The instrument is capable of magnifications up to 2,000,000x and is suitable for a range of applications, including materials science, biological research, and nanotechnology.
Automatically generated - may contain errors
Lab products found in correlation
Uc6 ultramicrotome, by Leica (2 mentions)
Poly bed 812 resin, by Polysciences (2 mentions)
Normal mouse igg, by Merck Group (1 mentions)
Uranyl acetate, by Electron Microscopy Sciences (1 mentions)
Carbon film 300 mesh copper grids, by Electron Microscopy Sciences (1 mentions)
Tecnai f20, by Thermo Fisher Scientific (1 mentions)
Phenylthiourea ptu, by Merck Group (1 mentions)
Schneider s insect medium, by Merck Group (1 mentions)
6 protocols using tecnai spirit 120
1
RNA Pol II CTD Immunoprecipitation and TEM
2
TEM Analysis of Protein Complexes
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TEM assays were performed for samples from co-IP assays using antibodies to RNA Pol II (CTD4H8) or FLI1 (Abcam, cat. no. 15289). Carbon Film 300 Mesh Copper grids (Electron Microscopy Sciences, cat. no. CF300-CU) were charged at 15 mA for 30 sec. Crosslinked immunoprecipitation samples were spotted onto charged grids and stained with 0.75% uranyl formate. For proteinase K-treated samples, the samples were treated with 5 µg of proteinase K and incubated for 30 min at 37°C before being spotted onto grids and stained with 0.75% uranyl formate. Images were collected from a FEI Tecnai Spirit 120S or FEI Tecnai F20 transmission electron microscope. Diameters of particles observed were measured with ImageJ software (U.S. National Institutes of Health, https://imagej.nih.gov/ij/ ).
3
Hemolymph Isolation and Imaging
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Third‐instar wandering larvae were bled in 50 μl of Schneider's insect medium (Sigma‐Aldrich) containing 1 μM phenylthiourea (PTU; Sigma‐Aldrich). The collected hemolymph was incubated on a glass coverslip for 1 h before being fixed for 2 h with 2% paraformaldehyde + 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Samples were then washed in cacodylate buffer (0.1 M, pH 7.4), fixed again in 1% osmium tetroxide and potassium ferrocyanide 1.5% in cacodylate buffer. After several washes in distilled water, samples were stained in 1% uranyl acetate in water, washed again, and then dehydrated in graded alcohol series (50, 70, 90, 95, 100%). Embedding was performed first in 1: 1 Hard EPON and ethanol 100%, and afterward in pure EPON, before being embedded on coated glass slides and placed at 60°C overnight. Images were acquired with a FEI Tecnai Spirit 120 kV (FEI Company, Eagle, The Netherlands).
4
Ultrastructural Analysis of iVDAC Mutant
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LLC-MK2 cultures in 25 cm2 flasks were infected with tachyzoites of iVDAC mutant and incubated (or not) with 0.7 µM ATc for 72 h. After that, infected cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and post-fixed for 45 min in the dark in 1% osmium tetroxide, 1.25% potassium ferrocyanide and 5 mM CaCl2, in 0.1 M sodium cacodylate buffer (pH 7.4). After post-fixation, infected cells were en bloc stained with uranyl acetate and lead aspartate. Then, samples were dehydrated in acetone solutions of increasing concentrations (30–100%) and embedded in PolyBed 812 resin (Polyscience, Warrington, PA) using flat-embedding moulds (EMS, Hatfield, PA). Ultrathin sections (70–80 nm) from three different blocks of −ATc and +ATc iVDAC were obtained in a Leica UC6 ultramicrotome and collected in 400 mesh copper grids (two grids per block). Sections were observed in a JEOL 1200 EX and FEI Tecnai Spirit 120 transmission electron microscope, and obtained images were analysed using ImageJ software. Images were acquired at Centro Nacional de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil.
5
Liposome Tubulation by ARF6 Protein
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All lipids were purchased from Avanti Polar lipids. Lipid mixtures, containing 40% phosphatidylcholine (DOPC), 30% phosphatidylethanolamine (DOPE), 20% phosphatidylserine, 8% phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and 2% cholesterol, were dried under nitrogen gas and then kept under vacuum for at least three hours. Dried lipid mixtures were resuspended in 50 mM HEPES, pH7.4, 50 mM NaCl for 30 minutes at 37°C, and then underwent freeze-thaw (from liquid nitrogen to 37°C) for 5 cycles, followed by extrusion through membrane filters of 0.2 um to generate liposomes of 200 nm diameter.
For liposome tubulation analysis, ARF6 protein (0.4mg/ml) was incubated with liposomes (0.5mg/ml) at room temperature. After 30 minutes, the incubation mixture was applied onto a glow-discharged carbon-coated EM grid and stained with uranyl acetate. EM grids were examined with a transmission electron microscope (FEI Tecnai Spirit 120) and the micrographs were recorded under the nominate magnification of 49,000X.
For liposome tubulation analysis, ARF6 protein (0.4mg/ml) was incubated with liposomes (0.5mg/ml) at room temperature. After 30 minutes, the incubation mixture was applied onto a glow-discharged carbon-coated EM grid and stained with uranyl acetate. EM grids were examined with a transmission electron microscope (FEI Tecnai Spirit 120) and the micrographs were recorded under the nominate magnification of 49,000X.
6
Ultrastructural Analysis of iVDAC Mutant
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LLC-MK 2 cultures in 25 cm 2 flasks were infected with tachyzoites of iVDAC mutant and incubated (or not) with 0.7 µM ATc for 72 hours. After that, infected cells were fixed with 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and post-fixed for 45 min in the dark in 1% osmium tetroxide, 1.25% potassium ferrocyanide and 5 mM CaCl 2 , in 0.1 M sodium cacodylate buffer (pH 7.4). After post-fixation, infected cells were en bloc stained with uranyl acetate and lead aspartate then samples were dehydrated in acetone solutions of increasing concentrations (30-100%) and embedded in PolyBed 812 resin (Polyscience Inc., Warrington, PA, USA) using flat-embedding molds (EMS, Hatfield, PA).
Ultrathin sections (70-80 nm) from three different blocks of -ATc and +ATc iVDAC were obtained in a Leica UC6 ultramicrotome and collected in 400 mesh copper grids (2 grids per block). Sections were observed in a JEOL 1200 EX and FEI Tecnai Spirit 120 transmission electron microscope and obtained images were analysed using Image J software.
Ultrathin sections (70-80 nm) from three different blocks of -ATc and +ATc iVDAC were obtained in a Leica UC6 ultramicrotome and collected in 400 mesh copper grids (2 grids per block). Sections were observed in a JEOL 1200 EX and FEI Tecnai Spirit 120 transmission electron microscope and obtained images were analysed using Image J software.
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