S2R+ cells were plated on glass coverslips coated with concanavalin A (Sigma, St Louis, MO) and transfected to express fluorescently tagged MyoII and βH-Spec using Effectene (Qiagen), per manufacturer’s instructions. Lateral indentation experiments were conducted 2 days after transfection with a modified Catalyst AFM integrated with an Axio Observer fluorescence microscope (Zeiss). To determine the effect of a localized mechanical force on MyoII and βH-Spec localization, the cantilever (MLCT with a pyramidal tip, Bruker) was first brought into full contact, at around 50 nN setpoint force, with the glass surface on a cell-free area within 10 μm from a target cell. Next, the cell was laterally translated into the stationary cantilever using the piezoelectric XY stage and the NanoScope software (Bruker). The cantilever tip indented the edge of the cell by 2–5 μm. Cells were simultaneously imaged with a plan-apochromat 63x/1.4 NA oil immersion objective (Zeiss). Time lapse images were taken at 5 second intervals using the Micro-Manager software (http://micro-manager.org/wiki/Micro-Manager ).
Plan apochromat 63x 1.4 na oil immersion objective
Manufactured by Zeiss
Sourced in Germany
The Plan-apochromat 63x/1.4 NA oil immersion objective is a high-performance optical lens designed for advanced microscopy applications. It provides a magnification of 63x and a numerical aperture of 1.4, which allows for excellent resolution and light-gathering capabilities. The lens is optimized for use with oil immersion techniques to achieve optimal imaging performance.
Automatically generated - may contain errors
Lab products found in correlation
Concanavalin a (cona), by Merck Group (2 mentions)
Effectene, by Qiagen (2 mentions)
Nanoscope software, by Bruker (2 mentions)
Mlct with a pyramidal tip, by Bruker (2 mentions)
Lsm 710, by Zeiss (1 mentions)
Zen 2010, by Adobe (1 mentions)
Imagej, by Adobe (1 mentions)
Axio observer z1 inverted microscope, by Zeiss (1 mentions)
Zen 2 blue edition, by Zeiss (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Plan neofluar 10x 0.3 na objective, by Zeiss (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
6 protocols using plan apochromat 63x 1.4 na oil immersion objective
1
Studying Force-Induced Cytoskeletal Dynamics
2
Lysosomal Cargo Trafficking Regulation
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Cells were incubated with 25 µg/ml DQ-BSA Green in media at 37°C overnight, and chased for 2 h in normal culture media. For P/Q-type VGCC blocker treatment, ω-agatonxin TK at different concentrations (50 nM, 100 nM, 250 nM, 500 nM, and 1 µM) or Bepridil hydrochloride (10 µM) were added to the culture together with DQ-BSA. The following steps were the same as described above. Individual coverslips were washed twice with PBS, and fixed (4% PFA, 15 min) cells were then stained with anti-Lamp1 and DAPI.
All the mounted samples were examined and imaged on a confocal microscope LSM710 (Carl Zeiss) outfitted with a Plan‐Apochromat 63X 1.4NA oil immersion objective (Carl Zeiss). Data were collected using Carl Zeiss software ZEN 2010 and processed in Image J and Photoshop CS (Adobe).
All the mounted samples were examined and imaged on a confocal microscope LSM710 (Carl Zeiss) outfitted with a Plan‐Apochromat 63X 1.4NA oil immersion objective (Carl Zeiss). Data were collected using Carl Zeiss software ZEN 2010 and processed in Image J and Photoshop CS (Adobe).
3
Microscopic Imaging with Apotome 2
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The Ludin chamber loaded with the sample was mounted on an Axio observer Z1 inverted microscope (Zeiss, Germany) equipped with an Apotome 2 module. The image acquisition was performed using a plan-apochromat 63x/1.4 NA oil immersion objective and excitation/emission filters of GFP and DsRed (Zeiss, Germany). Zen 2 blue edition was used for image acquisition (Zeiss, Germany) software. The images were obtained using an Axiocam 506 mono 6 Mp CCD camera with a frame size of 2752p x 2208p (as czi or tif files) at 8-bit depth. The image processing was done using Fiji (NIH, Bethesda, USA) and MetaMorph (Molecular Devices, San Jose, USA).
4
Confocal Imaging of Fluorescent Samples
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Confocal images were captured on a Zeiss LSM 510 Meta confocal microscope implemented on an upright Axioplan 2 microscope using Zeiss LSM 510 software. The 488 nm (15% intensity), 543 nm (80–100% intensity) and 633 nm (10–20% intensity) laser lines were used for excitation. Bandpass filters of 505–530 nm, 560–615 nm, and a longpass filter 650 nm were used to capture separate emission channels. A Zeiss Plan-Neofluar 10X/0.3 NA objective was used to capture low-magnification images and high magnification images were obtained using a Zeiss Plan-Apochromat 63X/1.4 NA oil-immersion objective. Confocal stack micrographs were prepared for publication using Volocity software (Perkin Elmer, Waltham, MA, United States). Adobe Photoshop 7.0.1 (Adobe Systems Incorporated, San Jose, CA, United States) was used to compile figures.
5
Studying Force-Induced Cytoskeletal Dynamics
6
Confocal Imaging and Co-localization
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Z-stack images have been captured with a Zeiss LSM800 confocal microscope using a Plan-Apochromat 63x, 1.4NA oil-immersion objective (Carl Zeiss). Then, the focal plane showing the maximum fluorescence intensity of green and red channels was selected for the co-localization analysis, which was performed using the freeware ImageJ. In particular, a straight line was drawn in correspondence of the protein fluorescence spot and the peaks of green and red fluorescence intensities along the line were calculated using the Plot Profile function.
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