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Glass slides

Manufactured by Ibidi
Sourced in Germany

Glass slides are flat, transparent surfaces used in various laboratory applications. They provide a stable platform for examining and analyzing samples under microscopes. Glass slides are durable, easy to clean, and provide a clear viewing area for observing and documenting samples.

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8 protocols using glass slides

1

Quantifying Cellular Oxidative Stress

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The amount of reactive oxygen species (ROS) produced within the cell was measured using CellROX Deep Red Reagent for oxidative stress detection (Invitrogen, USA). The cells were cultured in glass slides (ibidi) coated with poly-L-lysine (Sigma) and treated with respective compounds. CellROX reagent was added to media, along with Hoesht 33342 dye (Invitrogen, USA). Samples were imaged using Ziess LSM900 Confocal Microscope. Images were analyzed using Fiji. Signal was quantified per field of view and normalized to Hoesht. Mitochondrial mass, Mitochondrial membrane potential, and Mitochondrial ROS were measured similarly using MitoTracker Deep Red, TMRM and MitoSOX dyes.
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2

Oxidative Stress Imaging in U87 and U251 Cells

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U87 MG and U251 MG cells were grown on glass slides (Ibidi, Martinsried, Germany) by plating at a density of 5 x 104 cells/ml. After 48 h of incubation, the cells were washed with PBS and immediately incubated with DHE fluorescent dye in PBS (5 μM) for 30 min at 37°C. Cells were then washed with PBS and fixed with PFA (4%) for 15 min. The cells were washed with PBS and kept at 4°C until analysis. The cells were observed using a confocal microscope (Zeiss LSM700), and the images obtained were analyzed by Image J. The quantification was performed by measuring the fluorescence of 10 ROI per image in triplicate. Channels used: DAPI (405 nm) and EthD (555 nm).
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3

In situ Proximity Ligation Assay for Protein-Protein Interactions

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For detection of protein-protein interactions, in situ PLA was performed. The components used (Sigma) were as follows: anti-mouse PLA plus probe, anti-rabbit PLA minus probe and Detection Reagents Orange. HUVEC or U87MG cells were grown on μ-Chamber 12 well on glass slides (Ibidi©, Martinsried, Germany). After reaching 80% confluence or after appropriate treatment of cells, the assay was performed according to the manufacturer’s instructions. Briefly, after fixation and blocking, cells were incubated with the primary antibodies: mouse anti-VEGF (1:250), rabbit anti-VEGF (1:250), rabbit anti-Flk-1 (1:250), mouse anti-Flk-1 (1:250), mouse anti-NCL (1:50), goat anti-RPTPβ/ζ (1:250) (all from Santa Cruz Biotechnology Inc.), mouse anti-ανβ3 (1:500; Merck Millipore), mouse anti-PTN (1:500; Abnova, Heidelberg, Germany) and mouse anti-RPTPβ/ζ (1:250; BD Biosciences). Subsequently, cells were incubated with secondary antibodies conjugated with oligonucleotides. After hybridization and ligation of the oligonucleotides, the DNA was amplified. A detection mixture detected the amplicons, resulting in red fluorescence signals. Nuclei were counterstained with Draq5; cells were mounted with Mowiol 4–88 and visualized at room temperature with Leica SP5 confocal microscope.
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4

Biofilm Growth and Visualization

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Biofilms were grown in flow-chambers with individual channel dimensions of 1 x 4 x 40 mm as described previously [65 (link)]. One hour after inoculation, with bacteria, the growth medium flow (0.2 mm/s corresponding to laminar flow with a Reynolds number of 0.02) was started. When required, eDNA in biofilms was stained with 1 μM ethidium bromide prior to microscopy. Tobramycin (10 μM) was added to the biofilm medium after 4 days of cultivation. After 24 h of tobramycin treatment, propidium iodide (10 μM) was added to the flow cells to visualize the dead cells via confocal laser scanning microscopy. P. aeruginosa PA14 and petA mutant biofilms were grown under static conditions over 48 h at 37°C on glass slides (Ibidi) incorporating 300 μl chambers. After 48 h incubation, spent medium was removed and the biofilm eDNA stained with YoYo-1 (40 μM).
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5

Immunofluorescence Imaging of Cultured Cells

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For immunofluorescence experiments, HKs were cultured directly on glass slides (Ibidi) until confluent, as described above. Live cells were labeled with monoclonal antibodies at a concentration of 10 μg/ml for 30 min at 4°C. Cells were then fixed on ice using 3.7% paraformaldehyde for 10 min followed by permeabilization in 0.1% triton for 10 min at room temperature. Goat anti-human IgG cross-adsorbed secondary (Life Technologies) was then used prior to mounting with prolong gold containing Dapi (Life Technologies). Widefield images were acquired using a DMRXA2 microscope (Leica, Wetzler, Germany) equipped with a 63X/1.40 NA oil immersion objective. Images were acquired with an ORCA digital camera (Hamamatsu Photonics, Bridgewater, NJ). Analysis was preformed using Fiji ImageJ.
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6

Immunofluorescence Imaging of Cultured Cells

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For immunofluorescence experiments, HKs were cultured directly on glass slides (Ibidi) until confluent, as described above. Live cells were labeled with monoclonal antibodies at a concentration of 10 μg/ml for 30 min at 4°C. Cells were then fixed on ice using 3.7% paraformaldehyde for 10 min followed by permeabilization in 0.1% triton for 10 min at room temperature. Goat anti-human IgG cross-adsorbed secondary (Life Technologies) was then used prior to mounting with prolong gold containing Dapi (Life Technologies). Widefield images were acquired using a DMRXA2 microscope (Leica, Wetzler, Germany) equipped with a 63X/1.40 NA oil immersion objective. Images were acquired with an ORCA digital camera (Hamamatsu Photonics, Bridgewater, NJ). Analysis was preformed using Fiji ImageJ.
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7

Imaging T Cell Activation Dynamics

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Glass slides (Ibidi, Germany) were washed with boiling piranha solution (70% of pure sulfuric acid with 30% of a 30% hydrogen peroxide solution) for 30 min. After intensive washing with PBS, Glass slides were functionalized with 5 μg/ml of anti-CD3 (OKT3) (eBiosciences, Thermo Fisher Scientific) or 5 μg/ml of IgG or 100 μg/ml of poly-L-lysine for 30 min at room temperature followed by extensive rinsing with PBS + 0.1% of BSA. A total of 8 × 105 electroporated T cells were washed and resuspended in PBS + 0.1% of BSA, then incubated on each substrate and fixed with 4% of paraformaldehyde during 20 min (Sigma, Germany) after 2, 4, 8, or 16 min after incubation. Cells were washed with PBS + 0.1% of BSA and incubated in 50 mM of NH4Cl (Sigma, Germany) for 20 min and then rinsed again with PBS + 0.1% of BSA. Cells were permeabilized with 0.5% of Triton-X 100 in PBS + 0.1% of BSA during 15 min before being rinsed with PBS + 0.1% of BSA. Cells were then labeled with 0.1 μg/ml of Phospho-ZAP70/Syk (Tyr319, Tyr352) Monoclonal Antibody (n3kobu5) PE (eBioscience, Thermo Fisher Scientific) in PBS + 0.1% of BSA + 0.05% of saponin (Sigma, Germany) during 1 h. After extensive rinsing with PBS, cells were imaged using a confocal microscope (Zeiss LSM 880 AiryScan) with a 63× 1.4 NA objective.
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8

Studying Biofilm Growth and Tobramycin Response

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Biofilms were grown in flow-chambers with individual channel dimensions of 1 x 4 x 40 mm as described previously [66] (link). One hour after inoculation, with bacteria, the growth medium flow (0.2 mm/s corresponding to laminar flow with a Reynolds number of 0.02) was started. When required, eDNA in biofilms was stained with 1 µM ethidium bromide prior to microscopy. Tobramycin (10 µM) was added to the biofilm medium after 4 days of cultivation. After 24 h of tobramycin treatment, propidium iodide (10 µM) was added to the flow cells to visualize the dead cells via confocal laser scanning microscopy. P. aeruginosa PA14 and petA mutant biofilms were grown under static conditions over 48 h at 37 °C on glass slides (Ibidi) incorporating 300 µl chambers. After 48 h incubation, spent medium was removed and the biofilm eDNA stained with YoYo-1 (40 µM).
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