Polyacrylamide gels of variable stiffness were prepared on glass coverslips or 60 mm or 150 mm dishes with modifications of the method initially described28 (link), 29 (link) . Briefly, the glass coverslips or glass dishes were treated with 0.1M NaOH, followed by 0.5% 3-APTMS (Sigma) and 0.5% glutaraldehyde solution (Electron Microscopy Sciences) and air-dried. Acrylamide and bis-acrylamide (National Diagnostics) were mixed in defined ratios (determined by the stiffness) in PBS, and gel polymerization was promoted by the addition of 10% APS (1/100 volume, Millipore) and TEMED (3/1000 volume, Invitrogen). The gel mixture was then dropped on the coverslips and a coverslip that had been treated with a hydrophobic silicone polymer (Rain-X, Illinois Tool Works) was lowered onto the gel droplet. For glass dishes, waterproof membrane films were cut according to the dish size to cover the gel. After removing the top coverslips, the gels were washed with PBS and 0.05% sulfo-SANPAH (Covachem) was added. The gels were then exposed to UV for 5 mins (coverslips) or 20 mins (dishes) to facilitate cross-linker activation and incubated with 0.1mg/ml Collagen I (BD) in PBS for 2 hrs at room temperature. Excess Collagen I was washed off and the gels were kept in PBS at 4°C until cell seeding.
Glutaraldehyde solution
Manufactured by Electron Microscopy Sciences
Sourced in United States
Glutaraldehyde solution is a chemical fixative used in electron microscopy. It is a colorless, odorless liquid that functions as a cross-linking agent to preserve the structure of biological samples for analysis under an electron microscope.
Automatically generated - may contain errors
Lab products found in correlation
Temed, by Thermo Fisher Scientific (1 mentions)
3 aptms, by Merck Group (1 mentions)
Collagen 1, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Jem 2100 plus, by JEOL (1 mentions)
Sodium cacodylate buffer, by Merck Group (1 mentions)
Q150r es, by Quorum Technologies (1 mentions)
Hexamethyldisilazane hmds, by Merck Group (1 mentions)
Vega3 sem, by TESCAN (1 mentions)
Dulbecco s modified eagle s medium dmem f 12 medium, by Thermo Fisher Scientific (1 mentions)
Poly l lysine solution, by Merck Group (1 mentions)
4 protocols using glutaraldehyde solution
1
Polyacrylamide Gel Fabrication for Cell Culture
2
Transmission Electron Microscopy of Oocytes
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Human oocytes were counterstained with 6- or 12-nm gold-conjugated IgG secondary antibodies (Jackson ImmunoResearch, UK) for 1 hour, washed, and fixed with 3% glutaraldehyde solution (16220, Electron Microscopy Science, USA) in 0.1% BSA (A-1933, Sigma-Aldrich) in PBS for 1 hour at RT and 72 hours at 4°C. The samples were postfixed with 1% aqueous OsO4 (19152, Electron Microscopy Science, USA), embedded in 2% low-melting agarose type II (17856, Thermo Fisher Scientific), and dehydrated with a cold ethanol series on ice and a final dehydration with acetone-anhydride for 5 min. The samples were infiltrated using Epon Embed-812 resin (14120, Electron Microscopy Science, USA) at ratios 1:2, 1:1, and 2:1 with acetone for 5 to 30 min before polymerization at 60°C for 72 hours. The 80-nm-thick sections were examined at 120 kV using the transmission electron microscope Jeol JEM 2100-Plus by the TEM Centre imaging system.
3
SEM Analysis of DPSCs on Polymers
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Scanning electron microscopy (SEM) allows analyzing cell morphology and adhesion and the microstructural characteristics of the polymer surface. The DPSCs were observed after 24 h, 7, 14, 21, and 28 days of cultivation on the polymers. For each moment of analysis, the samples composed of DPSC, DPSC+ABS, and DPSC+PLA were fixed in 1.5 mL of glutaraldehyde solution (Electron Microscopy Sciences, Hatfield, PA, USA) in sodium cacodylate buffer (Sigma-Aldrich, St. Louis, MO, USA) and sucrose (Biotec-Labmaster, Pinhais, Brazil)) for 45 min. The fixing solution was removed, and the sample was washed with sodium cacodylate buffer and sucrose for 10 min. After fixation, the samples were dehydrated in a series of 10-min incubations with ethanol solutions (35%, 50%, 70%, 100%) (Biotec-Labmaster, Pinhais, Brazil) followed by Hexamethyldisilazane (HMDS) (Sigma-Aldrich, St. Louis, MO, USA) 100% for the same time. After drying, the metallization in gold was performed (QUORUM—Q150R ES), where they were covered with a thin gold deposition for SEM analysis (SEM Vega3, Tescan, Brno, Czech Republic).
4
Geltrex Gel Bed Protocol for Amniogenesis
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Geltrex gel bed was generated on the basis of a “sandwich” scheme developed recently for inducing amniogenesis from hPSCs (5 (link)). In brief, two 12-mm-diameter round glass coverslips were treated with air plasma (Harrick Plasma) for 2 min. One of the coverslips, which was to be coated with the gel bed, was soaked in poly-(l -lysine) solution (0.1 mg ml−1) (Sigma-Aldrich) for 30 min and then in 1% glutaraldehyde solution (Electron Microscopy Sciences) for another 30 min. The other coverslip was coated with poly-(l -lysine)-graft-poly-(ethylene glycol) (PLL-g-PEG, 0.1 mg ml−1; SuSoS) solution for 1 hour. To obtain gel beds with nominal thickness of 20, 60, and 100 μm, undiluted Geltrex (10, 30, and 50 μl, respectively) was then sandwiched between the two coverslips on ice before being incubated at 37°C for 30 min. The glass coverslip coated with the Geltrex gel bed was then gently separated from the PLL-g-PEG–coated coverslip before being submerged in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Thermo Fisher Scientific) and incubated at 37°C overnight before plating cells at the following day.
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