High resolution imaging datasets of cleared lungs were acquired on a Zeiss LSM 980 microscope using the Airyscan 2 SR-4Y mode with either a 40x/1.2 water immersion or a 63x/1.4 oil immersion objective. Raw Airyscan files were processed using Huygens Professional (Scientific Volume Imaging, Hilversum, Netherlands) and visualized in Imaris (Oxford Instruments). Thin section confocal images were acquired on a Zeiss LSM 980 microscope with a 40x/1.2 water immersion or a 63x/1.4 oil immersion objective and visualized in Imaris.
Lsm 980 microscope
Manufactured by Zeiss
Sourced in Germany
The ZEISS LSM 980 is a high-performance confocal laser scanning microscope. It features advanced optics and imaging capabilities to facilitate detailed observation and analysis of samples. The LSM 980 enables non-invasive, high-resolution imaging across a range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Ayriscan detector, by Zeiss (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Fv3000, by Olympus (1 mentions)
Qcm gelatin invadopodia assay, by Merck Group (1 mentions)
Cytopainter cell plasma membrane staining kit, by Abcam (1 mentions)
Duolink in situ red starter kit, by Merck Group (1 mentions)
Duolink blocking solution, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Ab145349, by Abcam (1 mentions)
Lsm 880 microscope, by Zeiss (1 mentions)
Rock2, by Abcam (1 mentions)
Fluorophore conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rock1, by Abcam (1 mentions)
Prock2, by Abcam (1 mentions)
Prock1, by Abcam (1 mentions)
Fgfr1, by Merck Group (1 mentions)
Mouse ve cadherin, by R&D Systems (1 mentions)
Affinipure fab fragment, by Jackson ImmunoResearch (1 mentions)
Antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
46 protocols using lsm 980 microscope
1
High-Resolution Lung Imaging Protocols
2
FRAP Analysis of Intron-tagged Proteins
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For FRAP experiments, cells harboring intron-tags in SMNDC1 and SRRM2 were seeded 24 h before imaging on a Zeiss LSM 980 microscope. 15 min before imaging, medium was changed to medium without phenol red containing DRAQ5TM 1:1000 to reduce autofluorescence and to mark nuclei. If cells were treated with compounds, these were added in the same step. After identifying a suitable cell, bleach and reference regions were defined. After taking one reference image, the bleach region was bleached 15 times for 5 milliseconds with 100% laser power at 488 nm for GFP and with 20% laser power at 546 nm for RFP. After bleaching, a new image was taken approximately every 3 s until 150 s after bleaching. Fluorescence intensities were quantified in the bleach and reference regions for every image and normalized to the intensity before bleaching.
3
Assessing Vascular Permeability in Lung Inflammation
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To measure vascular permeability in response to LPS challenge, dextran (70 kDa) and Evans blue dye (EBD) staining were used. In brief, the mice were injected with 50 µl of FITC-dextran or AF555-dextran (50 mg/ml) via tail vein 23.5 h after LPS injection. After 30 min, the whole lung tissues were harvested and perfused through the trachea with 200 μl of 50% OCT and immersed in 4% paraformaldehyde at 4°C for 8 h. Each lobe was embedded in OCT at -80°C for frozen sectioning. Then, 7-µm-thick sections were cut and stained with DAPI. The fluorescence of each group was measured by FV3000 (Olympus) and LSM980 microscope (Zeiss). The mice were injected via tail vein with 100 µl of 1% EBD 23 h after LPS injection. One hour later, the mice were sacrificed and the lung vasculature was flushed with 10 ml of PBS per mouse through the right ventricle to remove intravascular dye. The whole lung tissues were homogenized in 1.5 ml of formamide and incubated at 60°C for 48 h. The homogenates were centrifuged at 12000×g for 30 min, and the absorbance of the supernatant was measured at 620 nm in a 96-well plate.
4
Eugenol Effects on Leishmania Morphology
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Lmm promastigotes at 107 cells/mL were treated for 24 h with eugenol at concentrations between 0.5 and 30× IC50 (IC50 24 h = 5× IC50 72 h). The parasites were then centrifuged at 600× g for 10 min. The pellet was washed twice with PBS 1X, then fixed in 4% PFA. Then, 10 µL of the sample were mixed with 30 µL of a 10 µg/mL Nile Red solution in acetone and incubated for 10 min. The mixture was observed in a Zeiss LSM980 microscope. Quantification of Lmm morphology was obtained by measuring 4–6 images from two independent experiments for the Feret maximum diameter (accounting for length) and the Feret minimum diameter (accounting for width). The Feret ratio of Lmm was then calculated as follows:
Similarly, quantification of the Nile Red fluorescence was done by measuring the mean green intensity in 4–6 images per concentration of eugenol after manually drawing the Lmm contour using confocal images in black and white. The results were obtained from 2 independent experiments.
Similarly, quantification of the Nile Red fluorescence was done by measuring the mean green intensity in 4–6 images per concentration of eugenol after manually drawing the Lmm contour using confocal images in black and white. The results were obtained from 2 independent experiments.
5
Eugenol Inhibits Leishmania donovani Promastigotes
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Lmm promastigotes at the density of 107 cells/mL were treated for 24 h with eugenol at 1–30× IC50 (IC50 24 h = 5× IC50 72 h). Treated parasites were centrifuged at 600× g for 10 min. After washing twice with PBS, Lmm promastigotes were resuspended in 1 mL of PBS. Cells at 3 × 107 Lmm/mL were then transferred to a 24-well poly-l-lysine-coated plate. After several washes in PBS 1X, they were fixed with 4% formaldehyde in 0.1 M phosphate buffer for 20 min at room temperature. Permeabilization was done in 0.1% Triton-X100 for 5 min at room temperature. After several washes in Q-PBS (1% bovine serum albumin, lysine at 1 mg/mL, 0.01% saponin, and 0.025% azide in PBS 1X), the cells were incubated with anti-TbVP1 primary antibodies [50 (link)] for 1 h and then with fluorescent Alexa Fluor 488 goat antiguinea pig secondary antibodies for 1 h. The coverslips were mounted in prolong gold and left to polymerize for 24 h. The images were taken on a Zeiss LSM980 microscope. Quantification was obtained by measuring the mean green intensity of Lmm in 4–6 images per concentration of eugenol after manually drawing the Lmm contour using confocal images in black and white. The results were obtained from 1 experiment.
6
Quantitative Gelatin Invadopodia Assay
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Coverslips were inverted on 200 μL drop of QCM Gelatin Invadopodia Assay (Millipore) and heated to 37 °C. Coverslips were fixed in 0.5% glutaraldehyde for 15 min at 4 °C and after washing with PBS. Slides were sterilized with 70% ethanol and left in complete growth media for 1 h before use. Cells were cultured on gelatin-coated coverslips in a 24-well plate and left to adhere. Cells were then incubated for 48 hr in different experimental conditions and finally fixed and stained for CLSM examinations. Fluorescence signals were analyzed by Zeiss LSM980 Microscope equipped with a 63× oil objective. 3D reconstruction images of selected regions of interest (ROI) with evident matrix degradation spots were shown. To quantify invadopodia activity, black-and-white images of gelatin degradation are analyzed using FIJI software. The fraction of degraded area was analyzed in this way: Analyze>Set Measurements>select “Area Fraction”. The “area fraction” value was normalized to the number of nuclei in each image as measured from the DAPI channel in the same field [49 (link)].
7
SARS-CoV-2 Cell-to-Cell Spread Assay
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Vero E6, Calu-3, or Caco-2 were seeded in a well of a 96-well plate. Confluent monolayers were infected with at an MOI of 0.001. After 1 h of infection at 37°C, the inoculum was removed and replaced by normal media. Supernatants and infected cell lysates were collected at different time points postinfection and virus titer were determined as described above.
To analyze cell-to-cell spread of SARS-CoV-2, the virus plaque area was determined. Vero E6 seeded in a well of a 24-well plate were infected with 30 PFU of SARS CoV 2. After 1 h of infection, the inoculum was removed and cells were overlaid with VTM supplemented with 1.5% carboxymethylcellulose and incubated at 37°C. For testing the effect of HS antagonist, cells were pretreated with 10 μM surfen or 1 mg/mL lactoferrin for 1 h at 37°C before infection, and infection was performed in the presence of surfen or lactoferrin. Finally, after 1 h, the virus inoculum was removed and monolayer was overlaid with VTM supplemented with 1.5% carboxymethylcellulose and 10 μM surfen or 1 mg/mL lactoferrin. Cells were fixed and stained as described above and plaque areas were quantified using a Zeiss LSM 980 Microscope. The plaque area was measured with help of ZEISS ZEN lite 3.0 (Blue edition) and plotted using GraphPad Prism.
To analyze cell-to-cell spread of SARS-CoV-2, the virus plaque area was determined. Vero E6 seeded in a well of a 24-well plate were infected with 30 PFU of SARS CoV 2. After 1 h of infection, the inoculum was removed and cells were overlaid with VTM supplemented with 1.5% carboxymethylcellulose and incubated at 37°C. For testing the effect of HS antagonist, cells were pretreated with 10 μM surfen or 1 mg/mL lactoferrin for 1 h at 37°C before infection, and infection was performed in the presence of surfen or lactoferrin. Finally, after 1 h, the virus inoculum was removed and monolayer was overlaid with VTM supplemented with 1.5% carboxymethylcellulose and 10 μM surfen or 1 mg/mL lactoferrin. Cells were fixed and stained as described above and plaque areas were quantified using a Zeiss LSM 980 Microscope. The plaque area was measured with help of ZEISS ZEN lite 3.0 (Blue edition) and plotted using GraphPad Prism.
8
Calu-3 Cells Imaging with IOH-NPs
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Calu-3 (epithelial human
lung cancer) cells were seeded onto black
96-well plates with optical bottom (Thermo Fisher) at a density of
5 × 10–4 cells/mL. After 24 h, the IOH-NPs
were carefully redispersed and added to the growth medium at varying
concentrations. At varying time-points after treatment, the cells
were fixed with 4% formaldehyde in phosphate-buffered saline (PBS)
for 10 min at room temperature, followed by washing with phosphate-buffered
saline (PBS). Nuclei were labeled with DAPI (5 ng/mL). Cell membranes
were stained using the CytoPainter Cell Plasma Membrane Staining Kit
(Abcam, United Kingdom) according to the manufacturer’s instructions.
Confocal three-dimensional images were acquired using a Zeiss LSM980
microscope in Airyscan super-resolution mode.
lung cancer) cells were seeded onto black
96-well plates with optical bottom (Thermo Fisher) at a density of
5 × 10–4 cells/mL. After 24 h, the IOH-NPs
were carefully redispersed and added to the growth medium at varying
concentrations. At varying time-points after treatment, the cells
were fixed with 4% formaldehyde in phosphate-buffered saline (PBS)
for 10 min at room temperature, followed by washing with phosphate-buffered
saline (PBS). Nuclei were labeled with DAPI (5 ng/mL). Cell membranes
were stained using the CytoPainter Cell Plasma Membrane Staining Kit
(Abcam, United Kingdom) according to the manufacturer’s instructions.
Confocal three-dimensional images were acquired using a Zeiss LSM980
microscope in Airyscan super-resolution mode.
9
Oxidative Stress and Mitochondrial Dynamics
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Cells were harvested by centrifugation and washed twice with fresh medium before incubation for 40 min at 30°C with 10 μM 2’,7’dichlorodihydrofluorescein diacetate (H2DCFDA) or 100 nM Mitotracker Red CMXROS. 10 μl of cell suspension were then immobilized onto a plate in the presence 1 mg/ml concanavaline and subjected to bright field and fluorescence microscopy. Cells were analyzed at 30°C on a Zeiss LSM980 microscope equipped with a 63X (NA 1.4) and appropriate filter sets for acquisition of DCF and MitoTracker Red fluorescence.
10
Measuring Protein Dynamics via FRAP
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FRAP experiments were performed37 (link): the Ronin-GFP expressing cells were imaged using the LSM980 microscope with Ayriscan detector (Zeiss) with 488nm laser. Bleaching was performed using the 488nm laser at 100% power and images were collected every 2.5 s. Fluorescence intensity was measured using FIJI. Values are reported relative to pre-bleaching time points.
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