Glass coverslip

Manufactured by Marienfeld

Sourced in Germany

Glass coverslips are thin, transparent glass sheets used in various laboratory applications. They serve as a support surface for samples, allowing for microscopic observation and analysis.

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Lab products found in correlation

57 protocols using glass coverslip

Immunostaining of N-protein Transfected and Orthohantavirus-infected Cells

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Cells were plated on glass coverslip (Marienfeld) in 24-well plates at 2x10⁴ cells per well, then, N-protein transfected cells or orthohantavirus-infected cells were treated at the indicated times. For the detection of intracellular components, cells were fixed for 15 min at room temperature with 3.7% formaldehyde (FA, Sigma-Aldrich). Fixed cells were blocked with glycine at 20 mM (Sigma-Aldrich) in phosphate buffered saline (PBS) for 15 min. Cells were permeabilized with 0.5% Triton X100 in PBS for 5 min and then washed with PBS + 0.05% Tween20 (PBS-T). Cells were then incubated with the primary antibodies diluted in PBS-T containing 1% bovine serum albumin (BSA, Cell Signaling), for 1 h. After washing in PBS-T, primary antibodies were detected with Ig species-specific secondary antibodies conjugated either to Alexa Fluor 488 or Alexa Fluor 555 dyes (ThermoFisher Scientific) diluted in PBS-T-BSA 1%, for 1 h. Cells were washed with PBS-T and mounted in Fluoromount DAPI-G (Southern Biotechnology).
In order to detect cell surface antigens, living cells were incubated with primary and thereafter secondary antibodies, both diluted in PBS containing 0.1% sodium azide and complemented with 2% FBS and washings were also performed in PBS with 0.1% sodium azide. Cells were fixed at the end of the reactions with PBS /FA 3.7%, then processed as above for immunostaining.

Gallo G., Kotlik P., Roingeard P., Monot M., Chevreux G., Ulrich R.G., Tordo N, & Ermonval M. (2022). Diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction. PLOS Neglected Tropical Diseases, 16(10), e0010844.

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Glass Coverslip Surface Preparation

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Glass coverslip (Marienfeld, 22x40 mm No 1.5) were cleaned as described in Young et al.32 (link). Briefly, they were washed sequentially with distilled water, ethanol, distilled water, isopropanol, distilled water, ethanol, distilled water and excess water was dried with a stream of nitrogen. For visualization, we used two platforms. We either plasma-bonded 500 µm wide, 90 µm deep polydimethylsiloxane (PDMS) microchannels fabricated using standard photolithography methods, or we deposited PDMS gaskets on the clean coverslips. PDMS gaskets were obtained using biopsy punches of 3 or 6 mm in diameter.

Talà L., Fineberg A., Kukura P, & Persat A. (2019). Pseudomonas aeruginosa orchestrates twitching motility by sequential control of type IV pili movements. Nature microbiology, 4(5), 774-780.

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Microfluidic Device Fabrication by Soft Lithography

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The microfluidic channels were patterned by soft lithography. First, SU-8-100 photoresist (MicroChem, USA) was spin-coated onto a silicon wafer, which was then baked at 95 °C for 1 hour. The coated photoresist was selectively exposed to UV light through a mask bearing the microfluidic pattern, after which the exposed photoresist was developed in propylene glycol monomethyl ether acetate (PGMEA) photoresist developer (MicroChem, USA). Poly (dimethylsiloxane) (PDMS) solution containing Sylgard 184 silicone elastomer base and curing agent (weight ratio, 10:1; Dow Corning, USA) was cured on the patterned wafer at 80 °C for 1 hour in a dry oven. Inlet and outlet ports of all channels, including an EC channel, two side channels, and four ECM channels, were then opened with a biopsy punch. After autoclaving this patterned PDMS (upper part) and a glass coverslip (bottom part; Paul Marienfeld, Germany), the channel side of the upper part was irreversibly bonded to the bottom part by oxygen plasma treatment (CUTE; Femtoscience, South Korea). Next, a 1-mg/mL poly-D-lysine (PDL; MW: 30,000–70,000; Sigma-Aldrich, USA) solution in distilled deionized water (DDW) was immediately pipetted into the bonded device, after which the device was placed in a humidified 37 °C incubator for 4 hours. After washing away the excess PDL, the device was dried at 80 °C in an oven for more than 24 hours.

Han S., Shin Y., Jeong H.E., Jeon J.S., Kamm R.D., Huh D., Sohn L.L, & Chung S. (2015). Constructive remodeling of a synthetic endothelial extracellular matrix. Scientific Reports, 5, 18290.

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Fabrication of Nanostructured Surfaces

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Nanostructured surfaces used to align EC monolayers were fabricated as previously described28 34 (link). Briefly, nanoscale ridge/groove structures (700 nm ridges, 350 nm grooves and 300 nm heights) on a poly(ethylene terephthalate) (PET) film were replicated to a glass coverslip with 18 mm diameter (Marienfeld) by UV-assisted capillary force lithography (CFL)64 . A UV-curable resin poly(urethane acrylate) (PUA) (Minuta Tech. Korea) was used for the replication.

Song K.H., Lee J., Park H., Kim H.M., Park J., Kwon K.W, & Doh J. (2016). Roles of endothelial A-type lamins in migration of T cells on and under endothelial layers. Scientific Reports, 6, 23412.

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TIRF Imaging of Protein-Microtubule Interactions

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For TIRF experiments, clean glass coverslips were used (thickness 0.13 mm, Marienfeld, Lauda-Königshofen, Germany). Cleaning was achieved similarly to the QCM sensor, except for the last step, where UV/ozone was replaced by a 20-min activation with 14 M NaOH. Finally, the coverslips were rinsed with ultrapure water and dried under an argon stream. A silicone insulator (P24742, Molecular Probes) was then glued in order to prepare several individual microwells. Then, 2 µL of SUV at 1 mg/ml in citrate buffer were adsorbed for 1 h. They were finally thoroughly rinsed with citrate and Tris-buffer.
The proteins in the MES-Tris buffer at pH 7.4 were injected at 7.4 µM for WT and 17.8 µM for DD in 3 separate injections by removing and adding 10 µL volumes with a period of 30 min between each injection. After the last injection of proteins, the proteins were left to adsorb for 1 h on the SLB. Then, they were rinsed 3 times with MES-Tris buffer containing 10 µM Taxol and left to adsorb for 30 min before the injection of microtubules (single injection at 25 µM). After 1 h of adsorption, they were rinsed with MES-Tris buffer.

Lubart Q., Vitet H., Dalonneau F., Le Roy A., Kowalski M., Lourdin M., Ebel C., Weidenhaupt M, & Picart C. (2018). Role of phosphorylation in Moesin interactions with Phosphatidylinositol-4,5-bis(phosphate)-containing biomimetic membranes: a biophysical study. Biophysical journal, 114(1), 98-112.

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Immunofluorescence Staining of Podocytes

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Mouse or human podocytes were seeded onto glass coverslips (Marienfeld) at 20 × 10³ cells/ml in 12-well plates and allowed to differentiate as described above. The cells were rinsed with ice-cold PBS and fixed with 4% PFA for 10 min at room temperature followed by permeabilization with 0.1% Triton X-100 for 5 min. After washing with PBS twice for 5 min each, the coverslips were incubated with blocking buffer in 5% donkey serum (Sigma-Aldrich, D9663) for 1 h at room temperature. For immunofluorescence staining, cells were incubated with custom rabbit anti-human D3 (1:100; Novus Biologicals, NBP1-05767; RRID:AB_1556282), and/or Mouse anti-human Synaptopodin (1:300; D-9; Santa Cruz Biotechnology, sc-515842), at 4°C overnight. The cells were washed with cold PBS and incubated with appropriate Alexa Fluor 488–labeled donkey anti-Rabbit IgG (1:1000; Molecular Probes, A-21206; RRID:AB_2535792) and/or Alexa Fluor 647–labeled donkey anti-mouse IgG (1:1000; Molecular Probes, A-31571; RRID:AB_162542) secondary antibodies at room temperature for 1 h. Cells were stained with 0.1 μg/ml DAPI (Invitrogen, D1306) in PBS. Then cells were examined using an LSM 700 laser scanning fluorescence confocal microscope with ZEN software (Zeiss).

Agarwal S., Koh K.H., Tardi N.J., Chen C., Dande R.R., WerneckdeCastro J.P., Sudhini Y.R., Luongo C., Salvatore D., Samelko B., Altintas M.M., Mangos S., Bianco A, & Reiser J. (2021). Deiodinase-3 is a thyrostat to regulate podocyte homeostasis. EBioMedicine, 72, 103617.

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Immunofluorescence Staining of Cells

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Immunofluorescence staining was performed as previously described (Mehlitz et al., 2010 (link)). Briefly, cells were seeded on glass cover slips of 15 mm in diameter (Paul Marienfeld, Lauda-Königshofen, Germany) and infected or re-infected for a respective time. After incubation, cells were fixed with 4% paraformaldehyde in PBS for 60 min. The fixed cells were stained using DAPI and an anti-SnGroEL primary antibody. Pictures were recorded using LEICA DMR microscope.

Koch R.D., Hörner E.M., Münch N., Maier E, & Kozjak-Pavlovic V. (2020). Modulation of Host Cell Death and Lysis Are Required for the Release of Simkania negevensis. Frontiers in Cellular and Infection Microbiology, 10, 594932.

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Differentiation of Neural Stem Cells

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Glass coverslips (12 mm in diameter; Marienfeld GmbH & Co. KG, Germany) were placed in 24-well culture plates (SPL, Seoul, Korea) and coated overnight with 15 mg/ml of polyornithine and 5 mg/ml of laminin (Sigma, MO, USA). Seven days after the primary culture, spheres were collected by centrifugation at 200 g, then dissociated with Accutase and mechanically disrupted. The resulting single cells were then washed to remove the enzyme, and seeded onto the coverslips at a density of 5 x 10 4 cells/well. The differentiation assay was done using appropriate differentiation medium for neuron (supplemented 1% penicillin-streptomycin, B27 supplement, and 2 mM glutamine), astrocyte (supplemented 1% penicillin-streptomycin, 1% fetal bovine serum, N2 supplement and 2 mM glutamine) and oligodendrocyte (supplemented 1% penicillin-streptomycin, B27 supplement, 2 mM glutamine and 30 ng/ml triiodo-L-thyronine) until day 14. The cultures were allowed to differentiate for 2 -3 weeks with medium change every 3 days in vitro, after which the differentiation into neurons, astrocytes, and oligodendrocytes was confirmed by immunocytochemistry.

Lee J.Y., Lee E.S., Kim S.P., Lee M.S., Phi J.H., Kim S.K., Hwang Y.I, & Wang K.C. (2017). Neurosphere formation potential resides not in the caudal cell mass, but in the secondary neural tube. The International journal of developmental biology, 61(8-9).

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Methacrylate Functionalization of Glass Coverslips

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Glass coverslips (Marienfeld, 170 ± 5 µm) were washed with isopropanol and acetone and dried using pressurized N₂. Subsequently, the surface was activated for 5 min by plasma treatment. The coverslips were immersed in a 4 × 10⁻³ m solution of 3‐(trimethoxysilyl)propyl methacrylate in toluene for 1.5 h. After washing twice in toluene and once in acetone, the methacrylate‐functionalized glass slides were used for TPLP microfabrication.

Weidinger B., Yang G., von Coelln N., Nirschl H., Wacker I., Tegeder P., Schröder R.R, & Blasco E. (2023). 3D Printing Hierarchically Nano‐Ordered Structures. Advanced Science, 10(28), 2302756.

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Immunotoxin Binding and Internalization Assay

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C4-2 cells were grown on glass cover slips (12 reaction fields, Paul Marienfeld, Lauda-Königshofen, Germany) for 24 h. Then the immunotoxin (20 μg/ml) was added and incubated for 4 h at 4°C for binding or at 37°C, 5% CO₂, for binding and internalization, respectively. For fixation, cells were washed and treated with 2% paraformaldehyde in PBS for 30 min at RT, washed again with 1% bovine serum albumin in PBS, and quenched for 10 min in 50 mM NH₄Cl in PBS. Mouse anti-human-c-myc mAb (BD Biosciences) and rabbit anti-human EEA1 Ab (Cell Signaling) were added and incubated for 45 min at RT. This was followed by a washing step and incubation with goat anti-mouse IgG-AF488 (Life Technologies, Darmstadt, Germany) and anti-rabbit IgG-DyLight®650 (Abcam, Cambridge, UK) for 45 min at room temperature. Slides were then washed extensively and mounted in Vectashield® containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc., CA, USA). Staining was analyzed with help of a Confocal Laser Scanning Microscope (TCS SP2 AOBS, Leica, Wetzlar, Germany; Leica LCS Confocal Software 2.6.1).

Michalska M., Schultze-Seemann S., Bogatyreva L., Hauschke D., Wetterauer U, & Wolf P. (2016). In vitro and in vivo effects of a recombinant anti-PSMA immunotoxin in combination with docetaxel against prostate cancer. Oncotarget, 7(16), 22531-22542.

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