GBM cells were grown on laminin-covered glass placed on 24-well plates under the same conditions described in Section 2.4 . To assess the cell expression of α7, α9, and muscle-type nAChRs, the cells were fixed with 4% PFA and then stained with Alexa-Fluor 555-conjugated α-bungarotoxin (50 nM, α-Bgt-Alexa 555, Thermo Fisher Scientific) overnight at 37 °C. The cells were washed extensively with extracellular buffer (140 mM NaCl, 2 mM CaCl2, 2.8 mM KCl, 4 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.4) to remove any unbound α-bungarotoxin. Controls were run simultaneously with 1 μM of unlabeled d-tubocurarine (Tocris). Twelve-bit digital images were obtained using a DuoScanMeta LSM510 laser scanning microscope (Carl Zeiss, Weimar, Germany) equipped with a Plan-Apochromat 63×/1.40 objective (numerical aperture). Image acquisition parameters were as follows: for green fluorescence—excitation at 488 nm and emission at 505–550 nm, for red fluorescence—excitation at 561 nm and emission at 575 nm. Pictures were processed with open-source ImageJ Fiji software version 1.54f.
Plan apochromat 63 1.40 objective
Manufactured by Zeiss
Sourced in Germany
The Plan-Apochromat 63×/1.40 objective is a high-performance microscope objective lens manufactured by Zeiss. It features a magnification of 63× and a numerical aperture of 1.40, providing excellent optical performance and resolution capabilities for a variety of microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Alexa fluor 555 conjugated α bungarotoxin, by Thermo Fisher Scientific (1 mentions)
D tubocurarine, by Bio-Techne (1 mentions)
Prime 95b scmos camera, by Teledyne (1 mentions)
Mounting medium, by Vector Laboratories (1 mentions)
Lsm software, by Zeiss (1 mentions)
Phycoerythrin conjugated secondary antibody, by Merck Group (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
3 protocols using plan apochromat 63 1.40 objective
1
Quantifying Nicotinic Receptor Expression in GBM Cells
2
Live-cell Imaging of Gene Silencing
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Prior to microscopy, cells were grown in YPD overnight at 30 °C, and then to log-phase in the same conditions. Then, 3 µL of 0.5 OD cells were then spotted onto CSM agar plates. Once the spots were dry, a sterile spatula was used to cut out a small square that encompassed the cell spots, which was then removed and inverted onto a 35 mm glass bottom dish (Thermo Scientific 150682). Cells were then imaged using a Zeiss ZA inverted fluorescence microscope with a Prime 95B sCMOS camera (Teledyne Photometrics), Plan-Apochromat 63×/1.40 objective (Zeiss), MS-2000 XYZ automated stage (Applied Scientific Instrumentation), and MicroManager imaging software (Open Imaging).
To generate timelapse images, samples were kept at 30 °C and humidified by a P-set 2000 Heading Incubation Insert (PeCon). Brightfield and fluorescence images were collected for 16 fields of view per sample, every 20 min for 10 h. A single Z-slice was acquired for each field of view at each timepoint. Images were analyzed with ImageJ (NIH). Silencing loss and silencing establishment events were counted manually with a single-blind approach.
To generate timelapse images, samples were kept at 30 °C and humidified by a P-set 2000 Heading Incubation Insert (PeCon). Brightfield and fluorescence images were collected for 16 fields of view per sample, every 20 min for 10 h. A single Z-slice was acquired for each field of view at each timepoint. Images were analyzed with ImageJ (NIH). Silencing loss and silencing establishment events were counted manually with a single-blind approach.
3
Immunofluorescence Analysis of GFP Expression
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HeLa cells were seeded (5 × 103 cells/well) into six-well cell culture dishes containing sterile coverslips, beadfected, and incubated for 48 h at 37 °C with 5% CO2. The coverslips were then washed with PBS (three 2-ml washes; Invitrogen, Paisley, UK) and fixed with 1% paraformaldehyde (20 min, 4 °C). Coverslips were again washed (three washes, each with 2 ml of PBS), permeabilized with 1% Trition X-100 in PBS for 30 min at room temperature (permeabilized samples only), and incubated with anti-GFP monoclonal antibody (Chemicon, Watford, UK) at 1:1000 dilution in PBS containing 4% w/v BSA with gentle rocking for 2 h at room temperature. Coverslips were washed as before and incubated with phycoerythrin-conjugated secondary antibody (Sigma, Poole, UK) (1:500 dilution in PBS containing 4% BSA) for 1 h with gentle rocking. Coverslips were washed and dried before being mounted in mounting medium (Vector Laboratories, Peterborough, UK). Slides were imaged with a Zeiss LSM 510 Meta confocal microscope using a Plan Apochromat ×63/1.40 objective mounted on an Axioplan 2 motorized upright stand, with images collected by LSM software (Zeiss). GFP fluorescence was detected using a Zeiss bandpass (505–550 nm) filter after excitation at 488 nm. Phycoerythrin fluorescence was detected using a long-pass 560-nm filter after excitation at 543 nm.
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