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6 protocols using conductive silver paint

1

Lipid Extraction and Microscopy Reagents

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LC-MS grade water (catalog no. W6–212) and chloroform (catalog no. C6704–4) were purchased from Fisher Scientific (Hampton, NH). Carboxymethylcellulose (CMC; catalog no. C4888) was purchased from Sigma-Aldrich (St. Louis, MO). Lipid standard D-lactosyl-ß-1,1′ N-palmitoyl-D-erythro-sphingosine (LacCer(d34:1), catalog no. 860576 P) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). SEM reagents glutaraldehyde (catalog no. 16020), paraformaldehyde (catalog no. 15710), sodium cacodylate buffer (catalog no. 11652), anhydrous ethanol (catalog no. 15055), and osmium tetroxide (catalog no. 19170) were all purchased from Electron Microscopy Sciences (Hatfield, PA). Conductive silver paint (catalog no.16062) was purchased from Ted Pella, Inc. (Redding, CA).
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2

Sample Preparation for SEM Imaging

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Samples for SEM analysis were subjected to gradual cooling (−20°C to −80°C) after fixation with 10% glutaraldehyde solution for 6–8 h. After the fixed samples were completely frozen, the samples were subjected to lyophilization for 48 h at about −105°C/100 mTorr vacuum. Freeze-dried samples were attached to an aluminum sample stub with conductive silver paint (Ted Pella, Inc., Redding, CA) and tightened using copper tape. Samples were then subjected to sputter-coating with a layer of ∼10 nm gold film before imaging by SEM. SEM images were acquired using an FEI Quanta 450 Scanning Electron Microscope at different magnifications. The accelerating voltage applied was 10 kV, and images were acquired using a dwell time of 30–60 μs.
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3

Synthesis and Characterization of CNT/GNS Nanocomposites

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The CNTs used in this study were multi-walled and were purchased from NanoIntegris (whose outer diameter is ~ 10 to 20 nm, and purity exceeds 95%). Latex solution was from Kynar Aquatec. Poly(sodium 4-styrenesulfonate) (PSS) (molecular weight is ~ 1 M) and N-methyl-2-pyrrolindinone (NMP) were acquired from Sigma–Aldrich. Low-defect few-layer GNS were synthesized from graphite microcrystalline powders (− 325 mesh, 99.995% pure, Alfa Aesar) using a surfactant-free, efficient, and economical LPE process8 (link),29 (link) by using a water-NMP (99% extra pure, Acors Organics) mixed solvent. EC was purchased from Sigma-Aldrich (viscosity 100 cP, 5% in toluene/ethanol 80:20, 48% ethoxyl). Conductive silver paint was purchased from Ted Pella for establishing electrodes on the fabricated nanocomposites. The substrates used were PET sheets of ~ 100 µm in thickness. Other chemicals and disposable laboratory supplies were from Fisher-Scientific.
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4

Scanning Electron Microscopy of Tracheal Cartilage

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Samples (two to three cartilage rings in length) from newborn piglet tracheas were fixed in modified Karnovsky's fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2) for 24 h at 4 °C. Postfixation was performed in 1% OsO4 at 4 °C three times with intervening 1% thiocarbohydrazide steps. The samples were dehydrated with increasing concentrations of ethanol followed by hexamethyldisilazane that was allowed to evaporate. Samples were mounted on aluminum specimen pin stubs (Cat# AGG301, Agar Scientific, Stansted, Essex, UK) with carbon tabs (Cat# AGG3347N, Agar Scientific, Stansted, Essex, UK) and conductive silver paint (Cat# 16040-30, Ted Pella, Redding, CA). To decrease charging, samples were sputter-coated with palladium before imaging at 3 kV in a field emission scanning electron microscope (Zeiss DSM 982 Gemini or Zeiss Leo Ultra 55, Carl Zeiss, Oberkochen, Germany).
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5

Ultrastructural Analysis of A. fumigatus Response to IRGB10 Peptides

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A. fumigatus was pre-grown 16 h before incubation with IRGB10 peptides for 5 h. Coverslips were fixed in a mixture of 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, Pennsylvania, USA, #16200), 2% paraformaldehyde (EMS #15700) in 0.1 M sodium cacodylate buffer (pH 7.4) (EMS #12310). Samples were then rinsed in 0.1 M sodium cacodylate buffer four times for five minutes each, followed by a secondary fixation in 1% osmium tetroxide (#19140) in ultrapure water for 60 minutes. The samples were then rinsed four times in ultrapure water for five minutes each, followed by dehydration in a graded ethanol series (30%, 50%, 70%, 90%, 100% ×2) for ten minutes each step. The coverslips were then loaded into a critical point drier (Autosamdri 931.GL, Tousimis, Maryland, USA) which was set to perform multiple liquid CO2 exchanges in three ten-minute ‘purge’ cycles with Stasis mode. Once dried, the coverslips were mounted on aluminum stubs using conductive carbon adhesive tabs (EMS #77825-06), with the edges being painted with conductive silver paint (Ted Pella, Redding, California, USA, #16062) to reduce charging, and sputter coated with ~8nm of iridium (Desk V, Denton Vacuum, New Jersey, USA). The samples were then loaded into the Teneo VS SEM (FEI-Thermo Fisher, Hillsboro, Oregon, USA) and imaged.
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6

Preparing Samples for SEM Imaging

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For scanning electronic microscopy (SEM), a cover glass (disc) was cleaned with acetone and coated with 3 nm gold/palladium (Leica CPD300). Using a conductive silver paint (Ted Pella), the disc was fixed on a stub. A 100 μL drop of the stock solution was put on the cover glass and let it dry. After drying, the sample was examined with a Hitachi Regulus 8220 scanning electron microscope.
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