Time-lapse imaging was performed using a Carl Zeiss LSM780 inverted microscope with a Plan-Apochromat 20×/0.8 at 37°C under 5% CO2. Imaging of fixed samples was performed using a Carl Zeiss LSM700 upright microscope with a Plan-Apochromat 20×/0.8. Live imaging was recorded with a frame rate of 10 min.
Plan apochromat 20 0
Manufactured by Zeiss
Sourced in Germany
The Plan-Apochromat 20x/0.8 is a high-performance objective lens designed for microscopy applications. It provides a large field of view and a high numerical aperture, delivering excellent optical performance with reduced aberrations.
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Lab products found in correlation
Lsm 800, by Zeiss (3 mentions)
Lsm 780, by Zeiss (3 mentions)
Axio imager m2 microscope, by Zeiss (2 mentions)
C apochromat 40 1.20 w korr m27, by Zeiss (2 mentions)
Te300, by Nikon (2 mentions)
Tunel assay, by Roche (2 mentions)
Zen 2011 imaging software, by Zeiss (2 mentions)
Plan neofluar 40 1.3 oil objective lens, by Zeiss (2 mentions)
Plan apochromat 63 1.4 oil dic m27, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 780 confocal microscope, by Zeiss (2 mentions)
Plan apochromat 100, by Zeiss (2 mentions)
Lsm 780 inverted microscope, by Zeiss (1 mentions)
Lsm700 upright microscope, by Zeiss (1 mentions)
Laser scanning confocal microscope lsm710, by Zeiss (1 mentions)
Dapi solution, by Merck Group (1 mentions)
Mounting medium with dapi, by Merck Group (1 mentions)
Lsm 510 meta microscope, by Zeiss (1 mentions)
Axiocam icc1 camera, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
27 protocols using plan apochromat 20 0
1
Time-lapse Imaging of Samples
2
Transient and Stable GFP:SIMK Expression
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Agrobacteria carrying 35S::GFP:SIMK construct were used for transient transformation of N. benthamiana leaves and for further stable transformation into alfalfa RSY L1 plants. Transgenic plants were regenerated through somatic embryogenesis and cultivated in the culture chamber at above‐described conditions. Fluorescence signals were observed in N. benthamiana epidermal leaf cells and in alfalfa plant organs using Confocal Laser Scanning Microscope LSM710 (Carl Zeiss, Germany) equipped with Plan‐Apochromat 20×/0.8 (Carl Zeiss, Germany), and Confocal Laser Scanning Microscope LSM880 with Airyscan (Carl Zeiss, Germany) equipped with Plan‐Apochromat 20×/0.8 (Carl Zeiss, Germany). Samples were imaged with 488 nm excitation laser line and appropriate detection range for GFP emission. Image post‐processing was done using ZEN 2010 software.
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Agrobacteria carrying 35S::GFP:SIMK construct were used for transient transformation of N. benthamiana leaves and for further stable transformation into alfalfa RSY L1 plants. Transgenic plants were regenerated through somatic embryogenesis and cultivated in the culture chamber at above‐described conditions. Fluorescence signals were observed in N. benthamiana epidermal leaf cells and in alfalfa plant organs using Confocal Laser Scanning Microscope LSM710 (Carl Zeiss, Germany) equipped with Plan‐Apochromat 20×/0.8 (Carl Zeiss, Germany), and Confocal Laser Scanning Microscope LSM880 with Airyscan (Carl Zeiss, Germany) equipped with Plan‐Apochromat 20×/0.8 (Carl Zeiss, Germany). Samples were imaged with 488 nm excitation laser line and appropriate detection range for GFP emission. Image post‐processing was done using ZEN 2010 software.
3
Transwell Assay for Cell Migration
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2.5 x 104 MDA-MB-231 and MCF7 cells suspended in 1 ml serum-free medium were loaded onto a upper 8 μm pore size chamber inserted in a 12-well cell culture plate. The lower chamber was filled with DMEM supplemented with 1 % FBS as a chemoattractant. The cells were allowed to adhere before being treated with 10 ng/ml TNF-α and then incubated for 24 h at 37 °C in 5 % CO2. After this incubation, the inserts were removed and the remaining non-migrating cells on the upper surface of the membrane were removed with a cotton swab. The cells that migrated to the lower surface of the membrane were fixed with 5 % paraformaldehyde (PFA) in PBS for 20 min at room temperature, washed with PBS and then treated with 0.5 % Triton X-100 for 5 min. Next, the cells were stained with DAPI solution (Mounting Medium with DAPI, Sigma-Aldrich) and examined using a Zeiss LSM 510meta microscope at 20× magnification (Plan Apochromat 20×/0,8). Pictures of five randomly chosen fields were taken and migrating cells were counted using ImageJ software. The average number of migrating cells for each condition was calculated and differences in the numbers of cells that migrated through the membrane were calculated.
4
Hippocampal Neuron Morphometry in Mice
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Mice were anesthetized and perfused with 2% glutaraldehyde in PBS. Brains were removed and kept in 0.12 M phosphate buffer/2% glutaraldehyde. After treatment with osmium tetroxide, brains were embedded in Epon. Semithin (1 µm) coronal sections were cut from hippocampus, mounted with Eukitt, and observed using an Axio-Imager M2 microscope equipped with an AxioCamICc1 camera (Carl Zeiss). Images were acquired at 20× (Plan-Apochromat 20×/0.8) using AxioVision software (version 4.8.2.0; Carl Zeiss). The number of DG neurons was quantified by counting neuronal cell bodies in the whole DG area of the hippocampus. For EM analyses, ultrathin sections (70 nm) were cut and stained with uranium acetate and lead citrate (Almajan et al., 2012 (link)). Images were acquired using a transmission electron microscope at 13,500× (CM10; Philips) and an acceleration voltage of 80 kV using a camera (Gatan). Mitochondrial surface area was calculated from mitochondria imaged by transmission electron microscopy using ImageJ software (6 wk: Phb2fl/fl, n = 20; Phb2NKO, n = 31; Oma1ko/ko, n = 17; and Phb2NKOOma1ko/ko, n = 40; 14 wk: Phb2fl/fl, n = 20; Phb2NKO, n = 36; Oma1ko/ko, n = 26; and Phb2NKOOma1ko/ko, n = 43).
5
Immunofluorescence Staining of Frozen Tissue
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The frozen sections were stained using antibodies described in Table 1 . Before staining, the sections were fixed in acetone (−20°C) for 10 min, washed in phosphate buffered saline (PBS), and blocked using 12% bovine serum albumin (BSA) in PBS for 60 min at room temperature. After washing in PBS, the sections were incubated sequentially with antibodies at room temperature for 60 min, followed by washing 3× with PBS after staining. Sections were then mounted using Vectashield Vibrance Antifade Mounting Medium with 4′,6‐diamidino‐2‐phenylindole (DAPI, Vector Laboratories).
Images of the stained sections were acquired with a confocal laser scanning microscope (Zeiss LSM 800), using a Plan‐Apochromat 20×/0.8. All samples were imaged within 12 h after being mounted, and imaged using identical settings optimized to obtain maximum signal with minimum background. AF‐488 was excited at 488, and fluorescence was detected at 515–620 nm. AF‐647 was excited at 640 nm while emission was detected at 656–700 nm. DAPI was excited at 405 nm while detecting fluorescence at 400–515 nm.
Images of the stained sections were acquired with a confocal laser scanning microscope (Zeiss LSM 800), using a Plan‐Apochromat 20×/0.8. All samples were imaged within 12 h after being mounted, and imaged using identical settings optimized to obtain maximum signal with minimum background. AF‐488 was excited at 488, and fluorescence was detected at 515–620 nm. AF‐647 was excited at 640 nm while emission was detected at 656–700 nm. DAPI was excited at 405 nm while detecting fluorescence at 400–515 nm.
6
Confocal Microscopy Imaging Protocol
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Microscopy images were acquired at 23°C using confocal microscope Zeiss LSM 800 with the following lenses: Zeiss C-Apochromat 40×/1.20 W Korr M27 and 20× PlanApochromat 20/0.8. The microscopy samples were embedded with Coverslip High Precision 1.5 H±5 μm (Marienfeld-Superior, Lauda-Königshofen, Germany). Immersion medium Immersol W 2010 (ne=1.3339) and immersion oil Immersol 518 F (ne=1.518) were used. Images were analyzed with Fiji, including plug-ins and adapted scripts. Figure panels were finally assembled using Photoshop CC 2019. Acquisition software was Zen 2.3 (blue edition).
7
Confocal Imaging of Vibratome Sections
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Images were taken with a confocal microscope (LSM700, Carl Zeiss) equipped with 405, 488, and 555 nm lasers and using a 20× air objective (Plan Apochromat, 20×/0.8, #420650-9901-000) or a 40x water immersion objective (C-Apochromat, 40×/1.2, #421767-9970-000). Tiled image stacks of vibratome sections were taken at a resolution of 2048 by 2048 pixels and a step size of 0.89 µm. The tiled image stacks were subsequently stitched together using Zeiss ZEN black edition software. The contrast and brightness of the images was adjusted using Zen Black, Adobe Photoshop, or Imaris (Bitplane, Zurich, Switzerland) software.
8
Confocal Microscopy Imaging Protocol
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Microscopic images were acquired at 23°C on confocal microscopes Zeiss LSM 800 with the following lenses: Zeiss C-Apochromat 40×/1.20-W Korr M27, 20× PlanApochromat 20/0.8. The microscopic samples were embedded with Coverslip High Precision 1.5H ± 5 µm (Marienfeld-Superior). Immersion medium Immersol W 2010 (ne = 1.3339) and Immersion oil Immersol 518 F (ne = 1.518) were used, respectively. Images were analyzed with Fiji including plug-ins and adapted scripts. Figure panels were finally assembled by using Photoshop CS5. The acquisition software was Zen 2.3 (blue edition).
9
Live Imaging of Protein Localization
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A time-lapse analysis was performed using a Zeiss LSM-780 or 880 confocal microscope. The objective lens and time course are described in the text and figure legends. Fundamentally, we perform live imaging with 20× objective lens (Plan APOCHROMAT 20×/0.8, Zeiss) with large frame sizes (2,048 × 2,048 dpi), and magnify the point being observed. With an objective lens with higher magnification (63×, 100×), we lost track of the particles containing the tagged protein because of the shallow depth of the field.
10
Histological Analysis of Muscle Tissues
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Muscle tissues were prepared for histology as previously described (Sacco et al., 2005 (link)). Cells and muscle sections were fixed with 1.5% PFA, permeabilized in 0.1% Triton, and blocked in 20% goat serum. Incubation with the primary antibodies was performed overnight at 4°C. See Supplemental Experimental Procedures for a list of antibodies used. Cell death was measured by TUNEL assay (Roche). Images were acquired using an inverted epifluorescent microscope (Nikon TE300), CCD SPOT RT camera, and SPOT imaging software (Diagnostic Instruments) or a LSM170 laser-scanning confocal microscope, Plan-Apochromat 20×/0.8 or Plan NeoFluar 40×/1.3 oil objective lens, and ZEN 2011 imaging software (Zeiss). All images were composed and edited and modifications applied to the whole image using Photoshop CS6 (Adobe).
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