12 mm glass coverslips

Manufactured by Thermo Fisher Scientific

Sourced in United States

12 mm glass coverslips are thin, circular glass plates used in microscopy to cover and protect specimens on microscope slides. They provide a flat, transparent surface for observation and analysis of samples under a microscope.

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16 protocols using 12 mm glass coverslips

Visualizing HeLa Cell Membrane and ER Structure

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HeLa cells were reseeded 24 h post-transfection on 12 mm glass coverslips (Fisher Scientific). The next day, cells are fixed for 20 min with 4% EM-grade paraformaldehyde (PFA; Electron Microscopy Sciences 15710) and 4% sucrose in PBS at room temperature and washed three times in PBS.
To visualize the plasma membrane, cells were incubated with wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 (1:200, Invitrogen w11262) for 10 min at room temperature and washed twice with PBS. Afterwards, the nuclei were stained with Hoechst 33342 (1:2,000; Invitrogen H3570) for 10 min at room temperature and washed twice with PBS. Coverslips were mounted using fluorescent mounting medium (Dako S302380) and stored at 4 °C before being subjected to fluorescence microscopy.
To visualize the ER, fixed cells were permeabilized with 0.5% Triton-X in PBS for 5 min. After washing with PBS, coverslips were blocked with 3% BSA in PBS for 1 h at room temperature. Next, cells were incubated for 2 h with a primary chicken anti-calreticulin antibody as a marker for the ER (1:500, Ab14234, Abcam, USA), followed by three washes with PBS and staining with an Alexa Fluor 594® conjugated goat anti-chicken IgY secondary antibody (1:500, A-11042, Thermo Fisher Scientific, USA) for 1 h. Nuclear staining and mounting were performed as described above.

Bossaerts L., Hendrickx Van de Craen E., Cacace R., Asselbergh B, & Van Broeckhoven C. (2022). Rare missense mutations in ABCA7 might increase Alzheimer’s disease risk by plasma membrane exclusion. Acta Neuropathologica Communications, 10, 43.

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Isolation and Culture of Embryonic Rat Hippocampal Neurons

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Hippocampal neurons were isolated from embryonic rats at day 18 of gestation (E18) as previously described (Firestein et al., 1999 (link)). After isolation, the hippocampi were dissociated via manual trituration and plated at a density of 2 × 10⁵/well on 12-mm glass coverslips (Fisher) in 24 well plates (Corning). Coverslips were coated with 0.5 mg/ml poly-D-lysine (PDL; Sigma) for at least 1 h at 37°C prior to plating cells. Cultures were maintained in Neurobasal medium supplemented with B27, GlutaMAX, and penicillin/streptomycin (all from Life Technologies) in a humidified 37°C incubator with 5% CO₂. Cells were grown for 7 days in vitro (DIV) prior to transfection. This study was carried out in accordance with the recommendations of the Rutgers University Institutional Animal Care and Use Committee (IACUC). The protocol was approved by the Rutgers University IACUC.

O’Neill K.M., Donohue K.E., Omelchenko A, & Firestein B.L. (2018). The 3′ UTRs of Brain-Derived Neurotrophic Factor Transcripts Differentially Regulate the Dendritic Arbor. Frontiers in Cellular Neuroscience, 12, 60.

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Cryptococcus Internalization and Replication

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Mammalian host cells were infected with Cn at a multiplicity of infection (MOI) of 10, unless otherwise indicated. For experiments where microscopy was used to analyze the phagocytosis or intracellular replication of Cn, host cells were first cultivated on 12 mm glass coverslips (Fisherbrand) contained in 24 well plates (Falcon). Next, the host cells were infected and then incubated at 37°C in a 5% CO₂ atmosphere. At the indicated time points post-infection, culture media was removed, and the infected cells were extensively washed (6 to 8 times) with PBS, pH 7.4, before being processed for analysis of phagocytosis or intracellular replication. In some experiments, fluconazole protection assays were used to measure the phagocytosis and intracellular replication of the pathogen as previously described (Qin et al., 2011 (link)). For assays that measured pathogen internalization by immunofluorescence microscopy, infected cells were fixed and processed as described (Qin et al., 2011 (link)) without adding Triton X-100 to the staining buffer.

Pandey A., Ding S.L., Qin Q.M., Gupta R., Gomez G., Lin F., Feng X., da Costa L.F., Chaki S.P., Katepalli M., Case E., van Schaik E., Sidiq T., Khalaf O., Arenas A., Kobayashi K., Samuel J.E., Rivera G., Alaniz R.C., Sze S.H., Qian X., Brown W.J., Rice-Ficht A., Russell W., Ficht T.A, & de Figueiredo P. (2017). Global reprogramming of host kinase signaling in response to fungal infection. Cell host & microbe, 21(5), 637-649.e6.

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Dual Infection of Macrophages with Leishmania and Borrelia

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DH82 (2 × 10⁵/well) and RAW 264.7 (5 × 10⁴/well) cells were seeded in 24-well tissue culture-treated plates containing sterilized 12 mm glass coverslips (Fisherbrand, Waltham, MA, USA) and incubated at 37 °C, 5% CO₂ overnight, as described elsewhere [54 (link)]. The following day, cells were infected with L. infantum at 1:10 MOI. Two hours post-infection, wells were thoroughly washed with pre-warmed supplemented DMEM to remove non-internalized parasites and B. burgdorferi were inoculated at 1:25 MOI. 24 h and 48 h post coinfections, coverslips were fixed with methanol (Research Products International, Mount Prospect, IL, USA), stained with HEMA 3 (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Slides were assessed under a light microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan) at 1000× magnification by blinded reviewers to quantify percent L. infantum-infected cells and number of intracellular amastigotes per 100 cells.

Pessôa-Pereira D., Scorza B.M., Cyndari K.I., Beasley E.A, & Petersen C.A. (2023). Modulation of Macrophage Redox and Apoptotic Processes to Leishmania infantum during Coinfection with the Tick-Borne Bacteria Borrelia burgdorferi. Pathogens, 12(9), 1128.

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Erlotinib and Signaling Pathway Inhibitors

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One x 10⁷ cells per 10cm plate were treated with erlotinib, LPN, SFN and U0126 alone or in combination at the indicated times and concentrations in standard culture media. After treatment, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer, supplemented with protease inhibitorsand clarified by centrifugation (12.000 g, 10 min). Equal quantities of protein were separated by sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis and assayed by immunoblotting (IB) using the indicated antibodies. For immunofluorescence studies, cells were seeded on 12mm glass coverslips (Fisher Scientific, ON), fixed in 3% paraformaldehyde, permeabilized with 0.2% TritonX-100 (Fisher Scientific, ON), and blocked in 3% bovine serum albumin. Cells were subsequently stained with the indicated primary antibody and appropriate AlexaFluor 546 secondary antibody. Alexa 488 Phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) were used to stain F-actin and nuclei, respectively. Cell images were captured on a Leica TCS-SP2 inverted confocal microscope (Leica Microsystems, ON).

Nagaria T.S., Shi C., Leduc C., Hoskin V., Sikdar S., Sangrar W, & Greer P.A. (2017). Combined targeting of Raf and Mek synergistically inhibits tumorigenesis in triple negative breast cancer model systems. Oncotarget, 8(46), 80804-80819.

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Culturing HEK293 Cells Expressing Na v1.7

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Human embryonic kidney (HEK293) cells stably expressing human Na_v1.7 purchased from Millipore (CYL3011; Billerica, MA) were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) in an incubator. Cells were maintained in a humidified atmosphere at 37°C with 5% CO₂-enriched air. Cells were passaged every 2-3 days by brief trypsinization. Cells treated with 0.25% trypsin-EDTA (Thermo Fisher Scientific) were plated on 12 mm glass coverslips (Fisher Scientific, Pittsburgh, PA) in 35 mm cell culture dishes. The electrophysiological recording was conducted after 24 hours.

Kim R.E, & Choi J.S. (2023). Polysorbate 80 blocked a peripheral sodium channel, Nav1.7, and reduced neuronal excitability. Molecular Pain, 19, 17448069221150138.

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Measurement of Intracellular Calcium Dynamics in N18TG2 Cells

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Mouse N18TG2 neuroblastoma cells were cultured as described [16 (link)], maintained in complete media containing Dulbecco’s Modified Eagle’s Medium (DMEM): Ham’s F-12 (1:1) supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) and 10% heat-inactivated bovine serum. Cells were grown in 75-cm² flasks at 37 °C in a humidified atmosphere (5% CO₂), harvested at sub-confluency, and transferred to 12 mm glass coverslips (Fisher Scientific Co., Waltham, MA, USA). At 50–75% confluence, cells were loaded for 15 min with Fura-2 (5 µM) in Krebs–Henseleit Buffer (KHB) containing (in mM) NaCl 118, KCl 4.47, NaHCO₃ 25, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂·2H₂O 0.25, glucose 5.5. Cells were incubated in two different extracellular [Ca²⁺] (eCa²⁺) and the responses in intracellular [Ca²⁺] ([Ca²⁺]_i) were measured.

Pulgar V.M., Howlett A.C, & Eldeeb K. (2022). WIN55212-2 Modulates Intracellular Calcium via CB1 Receptor-Dependent and Independent Mechanisms in Neuroblastoma Cells. Cells, 11(19), 2947.

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Ciliogenesis Induction and Immunostaining

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Cells were plated on 12 mm glass coverslips (Fisherbrand) in 24-well plates and, upon confluency, incubated in serum starvation media containing 0% FBS for 48 h to induce ciliogenesis. Cells were then washed with PBS (2 × 1 min), fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, washed with PBS (3 × 1 min), permeabilized with 0.1% Triton X-100 for 10 min, washed with PBS with 0.3% Tween 20 (3 × 1 min), and blocked in 5% bovine serum albumin (BSA) with 0.3% Tween 20 for 30 min. Cells were then incubated for 1 h in primary antibody against Arl13b (1:300) or acetylated α-tubulin (1:400) in a wet chamber. Following several more washes in blocking buffer, cells were incubated in dye-conjugated secondary antibodies and Hoechst dye for 30 min, washed again, and mounted on a coverslide with ProLong gold antifade reagent (Invitrogen).

Lauring M.C., Zhu T., Luo W., Wu W., Yu F, & Toomre D. (2019). New software for automated cilia detection in cells (ACDC). Cilia, 8, 1.

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Visualizing Macrophage Infection Dynamics

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RAW264.7 murine macrophage-like cells were seeded onto 12-mm glass cover slips (Thermo Fisher Scientific, USA) in 24-well cell culture plates at a density of 5 × 10⁵ cells per well and incubated in DMEM supplemented with 10% FBS in the presence of 5% CO₂ for one day. RAW264.7 cells were co-infected with a 1:1 mix of a WT strain producing GFP (SK016) and ΔCSK29544_02616 strain producing mCherry (SK017). After incubation at 37 °C for 45 min, the medium was replaced with fresh DMEM containing gentamicin (100 μg/mL) and incubated for an additional 45 min. The coverslips were washed with PBS three times and treated with 4% paraformaldehyde for 10 min. Fixed cells were washed again with PBS and treated with a mixture of Hoechst (Thermo Fisher Scientific, USA) and CellMask Deep Red (Thermo Fisher Scientific, USA) for 30 min. After washing the coverslips with PBS, they were analyzed by DMi8 Microscope (Leica, Germany).

Kim S., Yoon H, & Ryu S. (2018). New virulence factor CSK29544_02616 as LpxA binding partner in Cronobacter sakazakii. Scientific Reports, 8, 835.

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Isolation and Culture of Mouse Dorsal Root Ganglia

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For each tissue preparation, mice were euthanized via induction of isoflurane briefly followed by decapitation. The spinal column was removed and bisected; lumbar DRG were removed and pooled, and subjected to enzymatic incubations as described [20 (link),57 ,84 ]. DRG tissue was incubated in papain (45 U in HBSS+H, Worthington) for 20 min at 37°C, rinsed, and incubated in collagenase (1.5 mg/mL in HBSS+H, Sigma-Aldrich) for 20 min. DRG were washed again with HBSS+H, then transferred to 1mL of DRG media, which consisted of Neurobasal A medium (Invitrogen) supplemented with 100 U/mL penicillin/streptomycin (Corning), 2 mM GlutaMAX (Life Technologies), 2% B27 (Gibco) and 5% fetal bovine serum (Gibco). DRG were then manually triturated with fire-polished Pasteur pipettes (VWR) and passed through a 40-μm filter (VWR). DRG were then plated onto poly-d-lysine/collagen (Sigma-Aldrich)-coated 12-mm glass coverslips (Thermo Fisher Scientific) and stored at 37°C and 5% CO₂ until testing.

Slivicki R.A., Yi J., Brings V.E., Huynh P.N, & Gereau RW I.V. (2021). The cannabinoid agonist CB-13 produces peripherally mediated analgesia in mice but elicits tolerance and signs of CNS activity with repeated dosing. Pain, 163(8), 1603-1621.

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