Endothelial-cell spheroids were generated as described previously (36 (link)–39 (link)). Briefly, HUVEC were suspended in culture medium containing 0.2% (w/v) carboxymethylcellulose (Sigma-Aldrich) and seeded in round-bottom 96-well plates (Greiner, Germany) to form spheroids. When siRNA transfection was performed, spheroids were formed 24 h after the transfection of HUVEC with Del-1 siRNA or control siRNA. The following day, spheroids were embedded into rat collagen I (BD Biosciences, Germany) containing gels; after gel polymerization, cells were treated with bFGF-containing medium (bFGF, 30 ng/mL; PeproTech, Hamburg, Germany). After 24 h, images were acquired using an Axiocam MR digital camera with an Axiovert 100M inverted microscope using as objective a Plan-NEOFLUAR (at 10x/0.30) and were processed using AxioVision Rel 4.5 digital imaging software (all from Carl Zeiss, Jena, Germany). In vitro capillary sprouting was quantified by measuring the cumulative sprout length of each spheroid using a computer-assisted microscope (AxioVision 4.5 software, Carl Zeiss) and the mean cumulative sprout length of 10 spheroids/condition was calculated.
Axiovert 100 m inverted microscope
Manufactured by Zeiss
Sourced in Germany
The Axiovert 100 M is an inverted microscope manufactured by Zeiss. It is designed for various applications in life sciences and materials research. The microscope features a stable stand, multiple illumination options, and a range of objectives to accommodate different specimen types and magnification requirements. The core function of the Axiovert 100 M is to provide high-quality optical imaging and observation of samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Lsm 510 confocal microscopy system, by Zeiss (3 mentions)
Plan apochromat oil immersion objective, by Zeiss (3 mentions)
Plan neofluar objective, by Zeiss (3 mentions)
Axiovision 4, by Zeiss (3 mentions)
μ dishes, by Ibidi (2 mentions)
X tremegene hp dna transfection reagent, by Merck Group (2 mentions)
Round bottom 96 well plate, by Greiner (1 mentions)
Rat collagen 1, by BD (1 mentions)
Axiocam mr digital camera, by Zeiss (1 mentions)
Carboxymethylcellulose, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Lab tek chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Draq5, by Biostatus (1 mentions)
Lipofectamine plus reagent, by Thermo Fisher Scientific (1 mentions)
Yo pro 1, by Thermo Fisher Scientific (1 mentions)
Axiocam mrc digital camera, by Zeiss (1 mentions)
Microdishes, by Ibidi (1 mentions)
12 well plate, by Greiner (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
12 protocols using axiovert 100 m inverted microscope
1
In Vitro Endothelial Cell Spheroid Assay
2
Gastrin-Induced Autophagy Visualization
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Cells (10 000 cells in 200 μl medium with 10% FBS) were seeded on Lab-Tek™ chambered coverglass with 8 wells (NUNC, Thermo Scientific) and left overnight. Cells were serum starved and treated with gastrin (10 nM) and Baf A1 (100 nM) for 4 h. Cells were fixed (4% paraformaldehyde in PBS) for 10 min, washed (PBS x 2) and permeabilized (ice-cold MeOH) for 10 min on ice and washed (PBS x 2). Cells were stained with Draq-5 (1:1000), (Biostatus, DR05500) for 7 min, washed and stored at 4 °C over night before confocal microscopy. The cells were immunostained after a 1 h blocking using 3% goat serum in PBS followed by incubation of the properly diluted primary antibodies in 1% goat serum in PBS. Unbound antibodies were removed by washing 5 times 5 min incubations in PBS before fluorescent dye labelled secondary antibodies were applied according to the species origin of the primary antibody. Confocal microscopy studies were performed with a Zeiss Axiovert 100-M inverted microscope equipped with an LSM 510 laser-scanning unit and a 1.4 numerical aperture × 63 Plan-Apochromat oil immersion objective. Laser power was typically 30% and the pinhole was set to 0.8–1.2 μm. Multitracking was used for dual color imaging at 488 nm and 647 nm.
3
PMA-Induced Dynamics in LNCaP Cells
4
Histological Analysis of Plant Tissues
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Microtomy‐based histological analysis was performed as described previously (Shafi et al., 2015 ). Briefly, fresh cuttings of stems and prickles were fixed in FAA [1:1:18—formaldehyde: glacial acetic acid (50%):ethanol] at room temperature (RT). Dehydration was performed in a series of tertiary butyl alcohol series and embedded in paraffin (58–60℃). Sections (20 µm) were cut using a Finesse microtome (Finesse, USA) and stained in safranin (0.1%) for 3 hr, followed by 30 s of counterstain by fast green (0.1%) in clove oil. Stained sections were mounted on a glass slide by DPX (a mixture of distyrene, plasticizer, and xylene) using coverslip and visualized under a fluorescence microscope (Zeiss Axiovert 100 M inverted microscope). Observed sections showed deposition for lignified and suberized cell walls, and tannins in red, whereas nonlignified walls of phloem and parenchyma were green in colour (Ma et al., 1993 ).
5
Confocal Microscopy Immunostaining Protocol
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Confocal microscopy was performed using an Axiovert 100 M inverted microscope equipped with an LSM 510 laser‐scanning unit and a 63 × 1.2 NA plan Apochromat objective (Carl Zeiss, Inc., Thornwood, NY, USA). 488‐ and 543‐nm laser lines were used to excite the green and the red fluorophores, respectively. Emitted light was collected through band pass filters of 505–550 nm and 560–615 nm, respectively. For immunostaining, cells were fixed with 4% paraformaldehyde for 20 min., permeabilized with 0.1% Triton X‐100 for 5 min., blocked with 5% normal goat serum for 30 min., stained with primary antibodies for 1 hr and secondary antibodies for 30 min., all at room temperature.
6
Yo-Pro-1 Apoptosis Detection in ECs
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Yo-Pro-1 (Molecular Probes) staining was used for microscopic detection of adherent apoptotic cells. Endothelial culture medium supplemented with 25 µg/mL Etoposide was used as a positive control for apoptosis. Briefly, after 24 h of incubation with melanoma CM or basal medium under hypoxia, detached cells were removed and ECs were incubated with 0.5 µM Yo-Pro-1 for 15 min at 37°C. Fluorescence was visualized using an Axiovert 100 M inverted microscope with a 10X/0.30 Plan-Neofluar objective (Carl Zeiss) and an ORCA II ER camera (Hamamatsu Photonics Systems).
7
Live-cell Imaging of PMA-induced Signaling
8
Endothelial Cell Culture and Hypoxia
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ECs were seeded at a density of 3 × 104 cells per well in 24-well cluster plates and grown to 60% confluence. After 24 h in endothelial culture medium, CM was added to the ECs, and incubations continued for 24–72 h under normoxic or hypoxic conditions. After 24–72 h, cells were analyzed microscopically using an Axiovert 100 M inverted microscope with a 10X/0.30 Plan-Neofluar objective (Carl Zeiss) and images were captured with an Axiocam MRC digital camera using AxioVision 4.5 software (Carl Zeiss B.V., Sliedrecht, NL).
9
Microscopic Analysis of Plant Stem Anatomy
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Confocal microscopy analysis was done as described earlier Gill et al. [14] . Stem of WT and transgenic lines were harvested and fixed in formalin, glacial acetic acid and 50% ethyl alcohol (FAA) (1∶1∶18) at room temperature. Samples were subsequently dehydrated in a tertiary butyl-alcohol series [27] ; embedded in paraffin (melting point 58–60°C) and 8–10 mm sections were cut using a Finsee microtome. Sections were stained with 1% safranin in water and with 4% fast green in clove oil for 4 h and for 30 s, respectively. These were mounted in Canada balsam and examined using Confocal Laser Scanning Microscope (Zeiss LSM510 Meta Gmbh, Germany) equipped with a Zeiss Axiovert 100 M inverted microscope. Lignin auto fluorescence was collected by excitation/emission wave- lengths 488/505 nm.
For SEM analysis, stem cross-sections were fixed in two steps: first with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer, pH 7.4 for 1 h and then with 1% OsO4 in 0.1 mol/l cacodylate buffer, pH 7.4 for 30 m. After critical point drying, the samples were sputter-coated with gold, and the coated samples were viewed with a Hitachi S-3400N field emission SEM using an accelerating voltage of 30 kV.
For SEM analysis, stem cross-sections were fixed in two steps: first with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer, pH 7.4 for 1 h and then with 1% OsO4 in 0.1 mol/l cacodylate buffer, pH 7.4 for 30 m. After critical point drying, the samples were sputter-coated with gold, and the coated samples were viewed with a Hitachi S-3400N field emission SEM using an accelerating voltage of 30 kV.
10
Visualizing PKCε Translocation in LNCaP Cells
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