Samples of non-infected cells and phiKZ-infected cells after 5, 10, 15, 20, 30, and 40 min of infection were chemically fixed using glutaraldehyde (2.5%) for 30 min at room temperature. Cells were collected by centrifugation at 5000× g at 4 °C. Then, cell pellets were washed twice with sterile PBS, postfixed in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) for 30 min at room temperature, and subjected to EMbed 812 Kit (Electron Microscopy Sciences, PA, USA) embedding, according to the manufacturer’s protocol with the replacement of the 100% Propylene Oxide with the 100% Acetone. Thin sections were cut with a diamond knife (Diatome, Nidau, Switzerland) on a ultramicrotome Ultratome III 8800 (LKB, Bromma, Sweden), transferred to nickel grids (400 mesh, Merck, Darmstadt, Germany), covered with collodion, 2% in Amyl Acetate (Electron Microscopy Sciences, PA, USA). Sections were contrasted with gadolinium triacetate (Uranyl Acetate Alternative, TedPella, Redding, CA, USA). Electron microscopy studies were performed using the Libra120 120 kV transmission electron microscope (CarlZeiss, Oberkochen, Germany) at magnification 8000–16,000×.
Amyl acetate
Manufactured by Electron Microscopy Sciences
Sourced in Germany
Amyl Acetate is a clear, colorless liquid with a fruity, banana-like odor. It is commonly used as a solvent in various applications, including the preparation of specimens for electron microscopy. Amyl Acetate serves as a clearing agent, facilitating the infiltration and embedding of samples, which is a critical step in the specimen preparation process for electron microscopy analysis.
Automatically generated - may contain errors
Lab products found in correlation
Diamond knife, by Electron Microscopy Sciences (1 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (1 mentions)
Embed 812 kit, by Electron Microscopy Sciences (1 mentions)
Uranyl acetate alternative, by Ted Pella (1 mentions)
Paclitaxel, by Cytoskeleton (1 mentions)
Unlabeled porcine tubulin, by Cytoskeleton (1 mentions)
Silica microspheres, by Spherotech (1 mentions)
Glass coverslip, by Thermo Fisher Scientific (1 mentions)
Gmpcpp, by Jena Biosciences (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by GoldBio (1 mentions)
Hepes, by GoldBio (1 mentions)
Potassium chloride (kcl), by Thermo Fisher Scientific (1 mentions)
Norland 65, by Norland Products (1 mentions)
Mini extruder, by Cytiva (1 mentions)
Plusone repel silane es, by GE Healthcare (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
D biotin, by Avidity (1 mentions)
3 protocols using amyl acetate
1
Ultrastructural Analysis of phiKZ Infection
2
Characterizing Tubulin Interaction Dynamics
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Unlabeled porcine tubulin and its labeled analogs, (TRITC and biotin), GTP and Paclitaxel were purchased from Cytoskeleton, Inc. Mouse monoclonal anti-6xHis tag antibody and rat tubulin antibody which recognizes the C-terminal tail of α tubulin were purchased from ABCAM. GMPCPP was purchased from Jena Biosciences, Germany. Streptavidin-coated polystyrene beads 1% w/v (0.82 μm in diameter) and silica microspheres 9,92 % solid w/v (5.0 μm in diameter) were purchased from Spherotech. Amyl acetate and 2% Colloidon in Amyl acetate were purchased from Electron Microscopy Sciences. Glass coverslips 22 × 45 × 1.5 mM were purchased from Fisher Scientific. Glucose oxidase from Aspergillous niger, aqueous solution of catalase from bovine liver, dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), ATP, MgCl2, and Subtilisin A Bacillus licheniformis were purchased from Sigma Aldrich. Mouse anti-tubulin b3 antibody which recognizes the C-terminal tail of β tubulin was purchased from Bio-Rad Laboratories. Except where noted, all experiments were carried out in BRB80 buffer (80 mM PIPES, 1 mM MgCl2, 1mM EGTA, 2 mM MgATP, pH 6.9, 184 mM ionic strength). A subset of single-molecule TIRF measurements were carried out in BRB12 buffer (12 mM PIPES, 1 mM MgCl2, 1mM EGTA, 2 mM MgATP, pH 6.9, 36 mM ionic strength). Detailed ionic strength calculations are included in SI Appendix .
3
Reconstitution of Microtubule Dynamics
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GTP and paclitaxel (Taxol) were purchased from Cytoskeleton (Denver, CO). Guanosine-5′-[(α,β)-methyleno]triphosphate, sodium salt (GMPCPP), the nonhydrolyzable analog of GTP, was purchased from Jena Bioscience (Jena, Germany). Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), ATP, and glucose were purchased from Sigma (St. Louis, MO). HEPES and dithiothreitol (DTT) were purchased from GoldBio (St Louis, MO). EGTA, MgCl2, and KCl were purchased from Thermo Fisher Scientific (Waltham, MA). Sterile collodion (nitrocellulose) 2% in amyl acetate and amyl acetate were purchased from Electron Microscopy Sciences (Hatfield, PA). d-Biotin was purchased from Avidity (Aurora, CO). Chloroform solutions of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl), mini extruder, Nuclepore track-etch membrane (50 nm) from Whatman, and filter support disks 10 mm from Whatman were purchased from Avanti Polar Lipids (Alabaster, AL). High vacuum grease, silicone elastomer base (184 Sylgard), and the corresponding elastomer curing agent were manufactured by Dow Corning (Midland, MI). 2% Dimethyldichlorosilane in octamethylcyclooctasilane (PlusOne Repel-Silane ES) was purchased by GE Healthcare (Chicago, IL). Optical adhesive Norland 65 was purchased from Norland Products (Cranbury, NJ).
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