An equal volume of the vesicle solution and 1% MC solution (osmolality-matched) were mixed to increase the outer phase viscosity in order to prevent drifting during imaging. Well chambers (sticky-Slide 8 Well; ibidi) and glass chambers that consist of cover-slips (Carl Roth) fixed to microscope slides (Carl Roth) by 4-layer parafilm were used for imaging. Both imaging chambers were passivated with a BSA solution (10 mg/mL) for 20 min before the insertion of the vesicles. The vesicles were imaged immediately after the production to record the formation of polar patterns using glass chambers sealed with vacuum grease (Bayer Silicones) and 60 min after the production to observe the developed polar patterns using well chambers. Leica Thunder Imaging System with an HCX PL APO 63×/1.40 oil immersion objective and Leica TCS SP5 confocal microscope coupling resonant scanner with an HCX PL APO 63×/1.40 CS2 oil immersion objective were used to image the vesicles. The equirectangular projections of a single vesicle were produced using the Map3-2D software39 (link). Defect identification was performed manually from confocal z-projection images using Fiji/ImageJ.
Microscope slides
Manufactured by Carl Roth
Sourced in Germany
Microscope slides are flat, transparent glass or plastic surfaces used to hold and support samples for microscopic observation. They provide a stable and standardized platform for placing specimens, enabling clear visualization and analysis under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam mrc, by Zeiss (2 mentions)
Tcs sp5, by Leica (1 mentions)
Caffeine, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Propan 2 ol, by Avantor (1 mentions)
N heptane, by Avantor (1 mentions)
C18 reversed phase hptlc lichrospher, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Acquiremp, by Refeyn (1 mentions)
Discovermp software, by Refeyn (1 mentions)
Twomp mass photometer, by Refeyn (1 mentions)
Dm2500 light microscope, by Leica (1 mentions)
Axiovision 3, by Zeiss (1 mentions)
Axioplan 2 imaging microscope, by Zeiss (1 mentions)
Sterile coverslips, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Avantor (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
7 protocols using microscope slides
1
Imaging Polar Patterns in Vesicles
2
Quantitative Analysis of Caffeine
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Propan-2-ol (analytical grade) and n-heptane (analytical grade) were purchased from VWR Chemicals (Radnor, PA, USA) and methanol (HPLC grade) was purchased from Fisher Chemicals (Hampton, NH, USA). Bidistilled water was freshly produced with a distillation apparatus from Heraeus-Quarzschmelze GmbH (Hanau, Germany) in the laboratory. Caffeine (> 99.9% chemical purity) and 13C3-Caffeine standard solution (1 mg/mL in methanol, 99 atom % 13C, > 99% chemical purity) were obtained from Merck KGaA (Darmstadt, Germany). The used HPTLC plates including C18 reversed-phase (RP)-HPTLC LiChrospher®, NP-HPTLC, and cyano (CN)-modified HPTLC were all provided by Merck KGaA (Darmstadt, Germany). Microscope slides were acquired from Carl Roth GmbH + Co. KG (Karlsruhe, Germany), and uncoated TLC glasses were used from MACHEREY–NAGEL (Düren, Germany). Red Bull, Coca-Cola, coffee, and black tea as Caffeine-containing beverages were obtained from local grocery stores.
3
Mass Photometry Characterization Protocol
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MP was performed using a TwoMP mass photometer (Refeyn Ltd, Oxford, UK). Data acquisition was performed using AcquireMP (Refeyn Ltd. v2.3). MP movies were recorded at 1 kHz, with exposure times varying between 0.6 and 0.9 ms, adjusted to maximize camera counts while avoiding saturation. Microscope slides (1.5 H, 24×50 mm, Carl Roth) and CultureWellTM Reusable Gaskets were cleaned with three consecutive rinsing steps of double-distilled H2O and 100% isopropanol and dried under a stream of pressurized air. For measurements, gaskets were assembled on coverslips and placed on the stage of the mass photometer with immersion oil. Assembled coverslips were held in place using magnets. For measurements, gasket wells were filled with 10 µL of 1× phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4) to enable focusing of the glass surface. After focusing, 10 µL sample were added, rapidly mixed while keeping the focus position stable and measurements started. MP contrast values were calibrated to molecular masses using an in-house standard. For each sample, three separate measurements were performed. The data were analyzed using the DiscoverMP software (Refeyn Ltd, v. 2022 R1). MP image analysis was done as described67 (link).
4
Histological Evaluation of Liver Samples
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Formalin-fixed, paraffin-embedded liver samples were cut into 4-µm-thick sections using a Thermo Scientific HM 340E microtome (Fisher Scientific, Schwerte, DE), mounted on either Thermo Scientific SuperFrost Ultra Plus slides (Fisher Scientific, Schwerte, DE) or microscope slides (Carl Roth, Karlsruhe, DE), and heat-treated (37°C, overnight). The sections were stained with hematoxylin and eosin (HE) and covered with a glass cover slip before examination in a blinded manner with a Leica DM 2500 light microscope (Leica, Wetzlar, Germany) using a semi-quantitative scoring system (1, lowest; 5 highest). Evaluation criteria included degenerative changes, necrosis, hepatocytic vacuolization, fat accumulation, congestion, and cellular infiltration.
5
Hydrophilic Surface Preparation for Flow Chambers
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Flow chambers (≈40 μL) that consist of coverslips (22 mm×22 mm; Carl Roth) fixed to microscope slides (25 mm×75 mm; Carl Roth) by three-layer parafilm were used for the assays. The coverslips were sonicated for 30 min in 3 M NaOH and rinsed with MilliQ H2O before being cleaned in piranha solution (2:1, H2SO4/H2O2) for 10 min to render the surface hydrophilic. The piranha-cleaned coverslips were then rinsed and stored in MilliQ H2O. The microscope slides were sonicated for 30 min in 2 wt% Hellmanex aqueous solution (Hellma) and rinsed with MilliQ H2O before being stored in ethanol. The coverslips and microscope slides were used within 72 h after cleaning.
6
Autoradiography Analysis of Plant Sections
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For the MAR studies, eight-days old plants were transferred into vials (three plants per each vial) which were filled with 15 mL of HA at a concentration of 50 mg·L−1 with specific radioactivity of ~0.7 mCi L−1. Twenty four hours later the plants were sectioned using standard paraffin embedding and serial sectioning at 15 μm thickness as described by Ruzin39 . Then sections were put on microscope slides (Roth, Karlsruhe, Germany) and treated with chloroform to remove paraffin. Obtained slides were then subjected to MAR analysis using the tritium-sensitive X-ray film Kodak Biomax (Kodak, USA) or dipped in undiluted NBT-3 film emulsion (Kodak, Rochester, NY) and stored at 4 °C in the dark for 105 days before developing. Time of exposition was determined in preliminary experiments (data not shown). Film or emulsion development, fixing, and washing were performed according to procedure recommended by the manufacturer. Slides with plant sections were imaged with a Zeiss Axioplan 2 imaging microscope (Zeiss, Germany) equipped with a Zeiss AxioCam MRc color video camera, and Zeiss Axiovision 3.1 software.
7
Quantitative Fluorescence Microscopy Assay
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Sterile coverslips (Thermo Fisher Scientific) were placed into 12 well plates and cell suspension was added. One day later, cells were treated with the indicated drugs for 20 -24 h. Cells were fixed using 4% PFA (VWR) for 20 minutes at room temperature. For antibody staining, cells were permeabilized with 0.25% Triton/PBS. After several washing steps, cells were blocked for 1 h with 5% goat serum (v/v) and 5% BSA (v/v) in PBS at room temperature. After overnight incubation with the primary antibody at 4 C, cells were incubated with fluorescently labeled secondary antibodies (1:250) for 1 h at room temperature in the dark. Antibodies were diluted in PBS with 5% goat serum (v/v) and 5% BSA (v/v). Finally, cover slips were gently transferred onto microscope slides (Carl Roth GmbH) and fixed with Vectashield mounting media containing DAPI solution (Biozol Diagnostica). Fluorescence images were acquired with a Leica TCS SP5 confocal microscope. For live cell imaging, cells were added into μ-slide with a glass bottom (Ibidi) and staining was performed as described. For automated microscopy cells were seeded into 384 well plates and subjected to fixation using the CybiWell Vario robotic system. Fluorescence images were acquired using InCell Analyzer 2200 microscope (GE Healthcare) at 20X magnification in three channels (DAPI, Cy3 and FITC) with four sites per well.
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