2% w/v methylcellulose (25 centipoise, Sigma Aldrich, Dorset, UK) was prepared as described in Griffiths et al.27 (link). Gold particles were prepared by citrate reduction according to Frens44 and stored at 4 °C. To make the calibration solution, freshly made colloid was mixed 1:1 with 2% methylcellulose. To remove citrate and sodium ions, the mix was centrifuged for 2 hours at 8000 × g at 4 °C and sedimented particles resuspended in fresh 1% methylcellulose. The calibration solution was stored at months at 4 °C. Nanodiscs were prepared as described35 (link), and liposomes according to Marius et al.41 (link). Standard plastic support films were made using 1% pioloform (Agar Scientific, Stansted, Essex, UK) and copper hexagonal 100-mesh or square 200 mesh grids applied to the film. Nanorods (10 nm thick) were obtained from Sigma Aldrich (Dorset, UK).
Pioloform
Manufactured by Agar Scientific
Sourced in United Kingdom
Pioloform is a high-quality polymer film used as a support material for transmission electron microscopy (TEM) specimens. It is designed to provide a stable and uniform platform for the samples, ensuring accurate and reliable imaging results.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium molybdate, by Merck Group (2 mentions)
Phosphotungstic acid, by Merck Group (2 mentions)
Sodium silicotungstate, by Merck Group (2 mentions)
Methylamine tungstate, by Merck Group (2 mentions)
Ruthenium red, by Merck Group (2 mentions)
Uranyl acetate, by Merck Group (2 mentions)
Sodium nitrate nano3, by Merck Group (2 mentions)
Potassium nitrate kno3, by Merck Group (2 mentions)
Mc 25 centipoise, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
E coli polar lipid extract, by Avanti Polar Lipids (2 mentions)
Methylcellulose, by Merck Group (1 mentions)
Jem 1400 transmission electron microscope, by JEOL (1 mentions)
Phosphotungstic acid, by TAAB (1 mentions)
Veleta ccd camera, by Olympus (1 mentions)
Item software, by Olympus (1 mentions)
Jem1010 transmission em, by JEOL (1 mentions)
Orius sc1000b, by Ametek (1 mentions)
Tecnai 12, by Thermo Fisher Scientific (1 mentions)
Jem 1400, by JEOL (1 mentions)
10 protocols using pioloform
1
Calibration Solution for Gold Nanoparticles
2
Visualizing Bacterial Morphology via EM
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Electron microscopy using colony lifts was performed to visualise the general morphological characteristics of selected isolates. In this technique, 400 mesh Copper EM grids (Agar Scientific, Stansted, UK), coated in-house with pioloform (Agar Scientific, Stansted, UK) and carbon were placed directly onto the surface of bacterial colonies growing on blood agar and light pressure applied. After 1–2 min resting on the colonies, the EM grids were carefully removed and placed (cell-side-down) onto a drop of 3% (w/v) paraformaldehyde (PFA) in PBS. After 15 min incubation on the PFA, the grid was removed, its surface washed twice with deionized water and the bacterial cells negatively stained with 1.5% (w/v) phosphotungstic acid (Taab Laboratories). After drying the EM grids were examined in a JEM-1400 transmission electron microscope (JEOL, Welwyn Garden City, UK) and images acquired using an AMT XR60 digital camera (Deben, Tillbury, UK).
3
Freeze Fracture Replica Preparation
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Freeze fracture replicas were produced using a previously described method (Tarusawa et al., 2009 (link)) with some modifications. Cells (2 × 105 cells/ml) were seeded onto polyethylene terephthalate filters with 0.4-µm pore size (#353090; Corning) and cultured for 6 d. The cells were washed with 0.1 M phosphate buffer (PB) and fixed with 2% glutaraldehyde in 0.1 M PB at 4°C overnight. After washing with 0.1 M PB, the samples were cryoprotected with 30% glycerol in 0.1 M PB at 4°C overnight, and then rapidly frozen in between two copper carriers using a high-pressure freezing machine (HPM010; BAL-TEC). The cells were then fractured by separation of the two carriers at −120°C and replicated by platinum (45° unidirectional from horizontal level, 2 nm thick) and carbon (20 nm thick) in a freeze-fracture replica machine (BAF060; BAL-TEC). The replicated materials were transferred to a solution containing kitchen bleach (50%) and incubated with shaking until cell debris was removed from the replicas. The replicas were washed twice with distilled water and picked up onto grids coated with Pioloform (Agar Scientific). The samples were observed with a JEM1010 transmission EM (JEOL) at 100 kV accelerating voltage. Images were captured with a Veleta CCD camera using iTEM software (Olympus Soft Imaging Solutions). All freeze-fracture images are presented apical-side up in the figures.
4
Lipid Extraction and Chemical Reagent Procurement
5
Lipid Extraction and Chemical Reagent Procurement
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Pioloform (polyvinyl butyral) was obtained from Agar Scientific (Essex, UK).
Octyl glucoside purchased from Melford Laboratories Ltd (Suffolk, UK). E. coli Polar Lipid extract was obtained from Avanti Polar Lipids (Alabaster, AL). HEPES, potassium nitrate (KNO3), ammonium molybdate, uranyl acetate, phosphotungstic acid, sodium silicotungstate, methylamine tungstate, ruthenium red, MC (25 centipoise) and sodium nitrate (NaNO3) were purchased from Sigma Aldrich (UK).
Octyl glucoside purchased from Melford Laboratories Ltd (Suffolk, UK). E. coli Polar Lipid extract was obtained from Avanti Polar Lipids (Alabaster, AL). HEPES, potassium nitrate (KNO3), ammonium molybdate, uranyl acetate, phosphotungstic acid, sodium silicotungstate, methylamine tungstate, ruthenium red, MC (25 centipoise) and sodium nitrate (NaNO3) were purchased from Sigma Aldrich (UK).
6
Autopsy-Derived Coronary Artery Samples
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Twelve coronary arteries were collected during autopsy of six patients (aged 47 to 86 years) who died from non-cardiac causes. Autopsy was performed 6 to 9 hours after death (Department of Pathology, Academic Hospital Maastricht, Maastricht). Tissue collection was approved by the Maastricht Pathology Tissue Collection committee [5 (link)]. The Medical Ethics Committee of the Maastricht University approved the study protocol and all subjects gave their informed consent in writing.
The tissue samples, placed in Tissue Tek (Sakura, Zoeterwoude, the Netherlands), were frozen in liquid nitrogen and stored at -80°C. Tissue samples were sectioned in 5 μm thick sections and collected on glass slides and stored at -20°C or collected on pre-dried Pioloform (Agar Scientific LTD, Stansted, UK) films of 100 nm for PIXE analysis.
The tissue samples, placed in Tissue Tek (Sakura, Zoeterwoude, the Netherlands), were frozen in liquid nitrogen and stored at -80°C. Tissue samples were sectioned in 5 μm thick sections and collected on glass slides and stored at -20°C or collected on pre-dried Pioloform (Agar Scientific LTD, Stansted, UK) films of 100 nm for PIXE analysis.
7
Staining Preparation for Electron Microscopy
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Ammonium molybdate, uranyl acetate, phosphotungstic acid, methylamine tungstate and ruthenium red were kept in glass-walled containers and stored in the dark. The pH value of stains was adjusted using NaOH and or HCl. Stains were centrifuged prior to use at 13,800 × g for 10 min to remove aggregates. The stain solutions were used at the following concentrations (w/v) and pH values respectively: ammonium molybdate 2% pH 6.3, uranyl acetate 3% pH 3 or 3.5, sodium phosphotungstate 1% pH 7.2, sodium silicotungstate pH 7.2 1%, methylamine tungstate 2% pH 7, and ruthenium red 1% pH 8.1. Standard plastic support films were made using 1% pioloform (Agar Scientific, Stansted, Essex, UK) and copper hexagonal 100 or 150-mesh grids applied to the film.
8
Transmission EM and Negative Staining of Protein-Lipid Interactions
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For transmission EM, cells grown on glass coverslips were fixed, osmicated, dehydrated in a graded ethanol series and acetone, and infiltrated gradually with epon (TAAB) as described by Puhka et al. (2007) (link). 60-nm-thick sections were cut parallel to the coverslip and poststained with uranyl acetate and lead citrate. Specimens were observed using a Tecnai 12 (FEI) microscope equipped with an Orius SC 1000B bottom-mounted charge-coupled device camera (Gatan) at acceleration voltage of 80 kV.
The negative-staining samples were prepared by mixing 2 µM protein with 500 µM unilamellar vesicles (with a diameter of 400 nm) in 20 mM Hepes buffer, pH 7.5, and 150 mM NaCl at RT for 5 min. The mixture was applied to the glow-discharged Pioloform (Agar Scientific)- and carbon-coated copper grids and stained with 3% uranyl acetate. At each step, excess solution was removed by filter paper. The membrane morphologies were examined with a JEM-1400 (Jeol) with a Orius SC 1000B bottom-mounted charge-coupled device camera (Gatan). The lipid composition of MIM used for EM was POPC:POPE:POPS:PI(4,5)P2:cardiolipin = 40:34:3:5:18. The final concentrations of FAM92A1 and lipid were 2 and 500 µM, respectively.
The negative-staining samples were prepared by mixing 2 µM protein with 500 µM unilamellar vesicles (with a diameter of 400 nm) in 20 mM Hepes buffer, pH 7.5, and 150 mM NaCl at RT for 5 min. The mixture was applied to the glow-discharged Pioloform (Agar Scientific)- and carbon-coated copper grids and stained with 3% uranyl acetate. At each step, excess solution was removed by filter paper. The membrane morphologies were examined with a JEM-1400 (Jeol) with a Orius SC 1000B bottom-mounted charge-coupled device camera (Gatan). The lipid composition of MIM used for EM was POPC:POPE:POPS:PI(4,5)P2:cardiolipin = 40:34:3:5:18. The final concentrations of FAM92A1 and lipid were 2 and 500 µM, respectively.
9
Freeze-Fracture Replica Preparation for TEM
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Freeze-fracture replicas were produced using a method described previously (Tarusawa et al., 2009 (link)) with some modifications. The prefixed cells on the filters with a 0.4-µm pore size (#353090; Corning) were prepared in the same way as the samples for ultrathin sections. After three washes with 0.1 M PB, the samples were cryoprotected with 30% glycerol in 0.1 M PB at 4°C overnight and then rapidly frozen in between two copper carriers by using a high-pressure freezing machine (HPM010; BAL-TEC, Balzers, Liechtenstein). The cells were then fractured by separation of the two carriers at –120°C and replicated by platinum (45° unidirectional from horizontal level, 2 nm thick) and carbon (20 nm thick) in a freeze-fracture replica machine (BAF060; BAL-TEC). The replicated materials were transferred to a solution containing kitchen bleach (50%) and incubated with shaking until the cell debris was removed from the replicas. The replicas were washed twice with distilled water and picked up onto grids coated with pioloform (Agar Scientific, Stansted, Essex, UK) for TEM observation. All freeze-fracture images were presented apical-side up in the figures.
10
Staining Preparation for Electron Microscopy
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