Coverslips were first sonicated at 100% ethanol for 20 minutes, followed by plasma cleaning with a plasma cleaner at HIGH for 5 minutes. The coverslips were then immersed in the 2% PlusOne bind-silane(17-1330-01) solution made in ethanol for 30 minutes at room temperature. After rinsing the coverslips with ethanol for several times, the coverslips were dried at 90°C for 30 minutes. Purified total RNA was mixed in 4% acrylamide/bis solution (1610147; Bio-Rad) with fresh 25mM VA-044 initiator (27776-21-2; Wako Chemical) and the solution was degassed for 10 minutes on ice. A 12mm square coverslip (470019-000; VWR) was functionalized with GelSlick (Lonza; 50640). 1uL of the RNA hydrogel solution was added to the bind-silane functionalized coverslip and was spread out using the GelSlick functionalized square coverslip. The thickness of the hydrogel formed can be controlled by manipulating the volume added. The polymerization happened in a humid hybridization at 37°C for 2 hours. After polymerization is complete, the coverslips were immersed in 2× SSC for an hour or more to facilitate the removal of the top coverslips. smFISH measurement was then performed according to standard protocol.
Gelslick
Manufactured by Lonza
Sourced in Switzerland
GelSlick is a lab equipment product that facilitates the handling and transfer of delicate gel samples. It provides a smooth, non-stick surface to minimize sample loss and damage during gel manipulation.
Automatically generated - may contain errors
Lab products found in correlation
Acrylamide bis solution, by Bio-Rad (2 mentions)
Va 044 initiator, by Fujifilm (2 mentions)
Bis acrylamide, by Sangon (2 mentions)
Sulfo sanpah, by Thermo Fisher Scientific (1 mentions)
Collagen 1, by Corning (1 mentions)
Triton x 100, by Merck Group (1 mentions)
N n n n tetramethylethylenediamine, by Merck Group (1 mentions)
Ammonium persulfate ap, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (1 mentions)
Imidazole, by Merck Group (1 mentions)
Sodium phosphate, by Merck Group (1 mentions)
Va 086, by Fujifilm (1 mentions)
Tbs t, by Cell Signaling Technology (1 mentions)
Methacryloxyethyl thiocarbamoyl rhodamine b, by Polysciences (1 mentions)
Pureproteome nickel magnetic microparticles, by Merck Group (1 mentions)
Su 8 3050, by MicroChem (1 mentions)
5 protocols using gelslick
1
Single-Molecule RNA Hydrogel Immobilization
2
Single-Molecule RNA Hydrogel Immobilization
3
Fabrication of Tunable Stiffness Substrates
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Substrates with different stiffness were prepared as described previously by adjusting the ratio of 40% acrylamide to 2% bis-acrylamide (w/v) (Sangon Biotech, China) (Chen et al., 2019 (link); Tian et al., 2019 (link)). Tetramethylethylenediamine (TEMED, Klamar, China) and 10% ammonium persulfate solution (APS, Sinopharm, China) were used as cross-linking reagent. Before polymerization, 200 μl mixture was added onto a glass slide pre-treated with gel slick (Lonza, Switzerland), and a piece of silicified cover slip was put upon the gel solution to make a sandwich. After 5 min, the substrates were treated with sulfo-SANPAH (Thermo Fisher, USA) and coated with 1 mg/mL collagen I (Corning, USA) to facilitate the cell adhesion. Finally, the substrate was sterilized by 75% alcohol and UV before cell culture.
4
Microfluidic Protein Purification Protocol
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Acrylamide/bis-acrylamide 30% (w/w) solution, ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), imidazole, sodium phosphate, sodium chloride, sodium hydroxide, sodium deoxycholate, sodium dodecyl sulfate, and Triton X-100 were obtained from Sigma-Aldrich. 10× Tris-glycine was obtained from Bio-Rad. Tris-HCl, pH 6.8 buffer was obtained from Teknova. PureProteome nickel magnetic microparticles, 10 μm, were obtained from Millipore-Sigma. TBST was obtained from Cell Signaling Technologies. Methacryloxyethyl thiocarbamoyl rhodamine B was obtained from Polysciences. VA-086 was obtained from Wako. N-[3-[(3-benzoylphenyl)formamido]propyl]methacrylamide (BPMA) was obtained from Pharm-Agra Laboratories. Silicon wafers were obtained from WaferNet. SU-8 developer and photoresist (SU-8 3050) were obtained from Microchem. Recombinant Protein G with His Tag was obtained from Abcam and labeled in-house with Alexa Fluor 647 NHS ester (Life Technologies). 0.5 μm rhodamine-microbeads (FluoSpheres) were obtained from Life Technologies. Gel-Slick was obtained from Lonza.
5
Polyacrylamide Hydrogel Fabrication and Functionalization
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Polyacrylamide (PA) hydrogels were manufactured as described before [17] (link) [18] (link). Briefly, the solutions of 1 kPa PA gel was prepared by mixing 6% acrylamide (Sangon Biotech, China) and 0.05% bis-acrylamide (Sangon Biotech, China), and the solutions of 20 kPa PA gel was prepared by mixing 8% acrylamide and 0.264% bis-acrylamide. Polymerization was initiated with 0.1% tetramethylethylenediamine (TEMED, Klamar, China) and 0.01% ammonium persulfate solution. To make a substrate suitable for each well in a 6-well culture dish, 200 µl of the final solution was dropped onto a glass slide pre-treated with gel slick (Lonza, Switzerland), and a silanized coverslip 30 mm in diameter was placed on the top of the solution. The PA gel yielded was 30 mm in diameter and 200 μm in thickness. After 10 min of polymerization, the glass slide was removed and the coverslip with the gel attached was treated with sulfo-SANPAH (ProteoChem, USA) under UV for 15 min and coated with 0.1 mg/ml rat collagen I (Corning, USA). Before cell seeding, the gels were sterilized by UV for 30 minutes and 75% ethanol.
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