The endoplasmic reticulum was visualized by staining with the endoplasmic reticulum (ER)-specific dye, ER Tracker™ Blue/White DPX, which is retained within the ER lumen, thus labeling the ER tubular network. The assay was performed according to the manufacturer’s instructions. Briefly, cells were seeded on the glass coverslips and cultured under respective conditions. After that, the cells were incubated for 30 min at 37 °C and 5% CO2 with 1 µM ER Tracker diluted in experimental conditions. Then, the stained cells were fixed with 4% formaldehyde for 10 min, washed and mounted using the Vectashield anti-fade reagent. Images were taken with the Zeiss LSM780, Inverted Axio Observer Z.1 with Plan Apochromat 63×/1.4 Oil DIC objectives. The images were processed using the Zen Blue 2.1 software (Carl Zeiss Microscopy).
Plan apochromat 63 1.4 oil dic
Manufactured by Zeiss
Sourced in Germany, United Kingdom
The Plan-Apochromat 63×/1.4 Oil DIC is a high-performance objective lens designed for microscopy applications. It offers a magnification of 63x and a numerical aperture of 1.4, providing excellent resolution and light gathering capabilities. The lens is optimized for use with oil immersion, and it incorporates differential interference contrast (DIC) optics to enhance contrast and detail in transparent samples.
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Lab products found in correlation
Lsm 780, by Zeiss (4 mentions)
Inverted axio observer z 1, by Zeiss (1 mentions)
Zen blue 2, by Zeiss (1 mentions)
Ps 1 axioobserver z1, by Zeiss (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Axio imager 2, by Zeiss (1 mentions)
Lsm 700 confocal laser scanning fluorescence microscope, by Zeiss (1 mentions)
Ec plan neofluar, by Zeiss (1 mentions)
Ec plan neofluar 40 0, by Zeiss (1 mentions)
Plan apochromat 63 1.4 oil objective, by Zeiss (1 mentions)
Lsm900 microscope, by Zeiss (1 mentions)
Ec plan neofluar 20 0, by Zeiss (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Zen 2012 black software, by Zeiss (1 mentions)
Plan apochromat 40 1.3 oil dic, by Zeiss (1 mentions)
Lsm 780 nlo confocal laser scanning microscope, by Zeiss (1 mentions)
8 protocols using plan apochromat 63 1.4 oil dic
1
Imaging Endoplasmic Reticulum Architecture
2
Super-Resolution Microscopy Imaging Protocol
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Cells were imaged using an Elyra PS.1 AxioObserver Z1 motorized inverted microscope with a EMCCD camera and Plan-Apochromat 63×/1.4 Oil DIC (differential interference contrast) M27 Elyra objective (Zeiss) with solid-state lasers of 488 nm and 561 nm as light sources. ZEN lite software was used for both acquisition of z stacks (5 phases and 3 rotations grating) and reconstruction. Quality of raw and reconstructed data was determined using the ImageJ plugin SIM check (Ball et al., 2015 (link)). Cells were prepared as for confocal imaging.
3
Standardized Cell Imaging Protocol
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For all microscopy experiments, cells were fixed using methanol and stained with DAPI, and images were acquired with a microscope (Axio Imager 2; Carl Zeiss) with a 63x objective (Plan-APOCHROMAT, 63×/1.4 Oil DIC; Carl Zeiss) and processed with Axio Vision 4.8 software (Carl Zeiss). A Z-stack of ~2 μm thickness, with single planes spaced by 0.3 μm, was acquired and maximum-intensity projections were generated. To compare signal intensities, all images were taken under the same exposure conditions and processing methods.
4
Evaluating Mitochondrial Dysfunction in Cancer Cells
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Briefly, cells (BxPC-3, 3×104 cells/well and KP-4, 2×104 cells/well) were seeded into CELLview 35-mm glass-bottom cell culture dishes with four compartments for 24 h. These cells were treated with a combination of lapatinib (10 µM) and FTY720 (5 µM) at 37°C for 1 or 4 h. JC-1 dye (5 µM; FUJIFILM Wako Pure Chemical Corporation) was added during the last 30 min at 37°C. Next, the cells were visualized using an LSM 700 confocal laser scanning fluorescence microscope (Zeiss GmbH) equipped with a Plan-Apochromat 63×/1.4 oil DIC (Zeiss GmbH). All images were acquired and processed using ZEN 2012. The object-based fluorescence intensity was measured using ImageJ software.
5
Microscopy of fungal spore morphology
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For microscopy, around 1× 104 spores were inoculated on thin LNA (1% agar 1.66 mM MgSO4, 5.4 µM ZnSO4, 2.6 µM MnSO4, 18.5 µM FeCl3, 13.4 mM KCl, 0.34 µM biotin, and 0.75 µM thiamin) and incubated for at least 2 h at 28°C. For conventional fluorescence images, a Zeiss AxioImager Z.1 microscope with either Plan-Apochromat 63×/1.4 Oil DIC, EC Plan-Neofluar 40×/0.75, EC Plan-Neofluar 20×/0.50, or EC Plan-Neofluar 10×/0.30 objective and an AxioCamMR was used. The images were taken using ZEN 2012 Blue Edition.
For confocal imaging, a Zeiss LSM900 microscope with Plan Apochromat 63×/1.4 Oil objective and Airyscan 2 detector in super resolution mode was used.
For confocal imaging, a Zeiss LSM900 microscope with Plan Apochromat 63×/1.4 Oil objective and Airyscan 2 detector in super resolution mode was used.
6
Monitoring Salmonella Localization by eGFP
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Salmonella transformed with the eGFP-pBAD plasmid (a gift from Michael Davidson, Addgene plasmid #54762; http://n2t.net/addgene:54762 ; RRID:Addgene_54762) was used to confirm intracellular localization. Images of infected cells were acquired every hour using a Zeiss LSM 780 laser scanning confocal microscope equipped with a Plan-Apochromat 40×/1.3 Oil DIC or Plan-Apochromat 63×/1.4 Oil DIC objective (Zeiss, Jena, Germany). Single mages and time-lapse series were acquired using the ZEN 2012 Black software (Zeiss). Differential interference contrast was used for visualization of living cells on a TPMT detector. GFP fluorescence in Salmonella was quantitated by excitation at 488 nm with the argon laser and an emission at 490–569 nm in the Channel mode.
7
Multiscale Imaging of Cellular Processes
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For RRS, images of the cells were obtained using an Andor spinning disk: Olympus IX 83 inverted microscope, equipped with a Yokaga CSU-X1 Spinning disk Unit and BOREALIS technology for homogeneous illumination. The acquisition software is IQ3.
For RNAFish, UDS, TCR-UDS, and IF of splicing complex after local damage, images of the cells were obtained using a Zeiss LSM 780 NLO confocal laser scanning microscope and the following objective: Plan-Apochromat ×63/1.4 oil DIC (Differential Interference Contrast) M27 or ×40/1.3 oil DIC. The acquisition software is ZEN.
PLA and IF associated with PLA have been performed on a Zeiss Z1 imager right using a ×40/0.75 dry objective. The acquisition software is Metavue.
Images of the cells for each experiment were obtained with the same microscopy system and constant acquisition parameters. All images were analyzed with ImageJ software. All experiments have been performed at least two times and are biological replicates.
Error bars represent the standard error of the mean of the biological replicates. Excel was used for statistical analysis and plotting of all the numerical data. Statistics were performed using a Student’s test to compare two different conditions (siMock vs. siRNA X or No UV vs. after irradiation) with the following parameters: two-tailed distribution and two-sample unequal variance (heteroscedastic).
For RNAFish, UDS, TCR-UDS, and IF of splicing complex after local damage, images of the cells were obtained using a Zeiss LSM 780 NLO confocal laser scanning microscope and the following objective: Plan-Apochromat ×63/1.4 oil DIC (Differential Interference Contrast) M27 or ×40/1.3 oil DIC. The acquisition software is ZEN.
PLA and IF associated with PLA have been performed on a Zeiss Z1 imager right using a ×40/0.75 dry objective. The acquisition software is Metavue.
Images of the cells for each experiment were obtained with the same microscopy system and constant acquisition parameters. All images were analyzed with ImageJ software. All experiments have been performed at least two times and are biological replicates.
Error bars represent the standard error of the mean of the biological replicates. Excel was used for statistical analysis and plotting of all the numerical data. Statistics were performed using a Student’s test to compare two different conditions (siMock vs. siRNA X or No UV vs. after irradiation) with the following parameters: two-tailed distribution and two-sample unequal variance (heteroscedastic).
8
Quantifying Membrane-Cytoskeleton Dynamics
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Samples were scanned using a Zeiss 710 confocal laser scanning microscope with a Zeiss Plan-APOCHROMAT 63×/1.4 Oil DIC (Cambridge, UK). 8-bit images were acquired from basal cell surface to apical cell surface at an interval of 0.3 µm. Laser power, gain and offset value were optimised based on pseudo-colour to achieve optimal brightness and to avoid photobleaching. In some cases, sequential scanning mode was employed to prevent crosstalk between two different fluorophores. Confocal z-stack images were shown using maximumintensity projection in Fiji (NIH) [20] . Syndecan-1 expression was determined by reading thresholded fluorescence intensity on each cell and presented as percentage normalised to the parallel control, i.e. pure medium [19, 21] . To quantify the actin cytoskeleton, stacked images were separated according to the curved membrane. Sections from the start of curvature were projected again to obtain actin cortex image. Measurement of the cortical intensity was performed in the same region as cell nucleus on each cell [22] . The intensity was then normalised with respect to the parallel control. Those sections beyond the curvature were restacked, cropped to individual cell size and then imported into FilaQuant for basal filament counting [19, 23] .
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