Live zebrafish larvae were anesthetized in tricaine (MS-222) and their median fin folds were fixed with 4% paraformaldehyde (PFA) in PBS O/N at 4°C. The images of fins were obtained using a BZ-X710 (Keyence) with 10× NA 0.45 PlanApo and 20× NA 0.45 PlanFluor objective (Nikon) and LSM 780 (Carl Zeiss) with 63× NA 1.40 Oil PlanApo objective (Carl Zeiss). Images of the cultured mesenchymal cells were acquired using LSM 780 (Carl Zeiss) with 63× NA 1.40 Oil PlanApo objective (Carl Zeiss). ZEN (Carl Zeiss) and FIJI were used as image software for z projections. Live cell imaging was performed on a LSM780 (Carl Zeiss) with 20× NA 0.8 PlanApo (Carl Zeiss). The green and red fluorescence signal was detected by GaAsP detectors with 488 and 561 nm laser for live cell imaging. The images were processed into videos using Imaris software (Bitplane).
Lsm 780
Manufactured by Zeiss
Sourced in Germany, United States, Japan, France, China, Canada, United Kingdom, Switzerland, Singapore, Italy, Panama, India
The LSM 780 is a laser scanning microscope developed by Zeiss. It is designed for high-resolution imaging and analysis of biological samples. The instrument utilizes advanced confocal technology to provide detailed, three-dimensional images of specimens.
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Lab products found in correlation
Lsm 880, by Zeiss (356 mentions)
Lsm 710, by Zeiss (271 mentions)
Lsm 700, by Zeiss (220 mentions)
Lsm 510, by Zeiss (145 mentions)
Zen software, by Zeiss (130 mentions)
Axio observer z1, by Zeiss (129 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (118 mentions)
Hoechst 33342, by Thermo Fisher Scientific (108 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (102 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (90 mentions)
Lsm 510 meta, by Zeiss (84 mentions)
Triton x 100, by Merck Group (73 mentions)
Vectashield, by Vector Laboratories (73 mentions)
Lsm 800, by Zeiss (60 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (52 mentions)
Zen 2012, by Zeiss (51 mentions)
Zen 2011, by Zeiss (49 mentions)
Plan apochromat, by Zeiss (46 mentions)
Zen black software, by Zeiss (44 mentions)
Zen 2010, by Zeiss (39 mentions)
3 264 protocols using lsm 780
1
Live Zebrafish Larvae Fin Imaging and Culture
2
Fluorescence Imaging and FRET Analysis
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Cell fluorescence and FRET imaging were conducted on a Zeiss LSM 780 laser scanning confocal microscope equipped with a 63× oil immersion objective lens (N.A. 1.4). A 488 nm line of gaseous argon laser could excite Alexa Fluor 488 fluorescence so as to collect a spectrum with a 493 to 630 nm band-pass filter. By using a 633 nm line of gaseous argon laser, Alexa Fluor 647 fluorescence was excited to collect a spectrum with a 640 nm long-pass filter. The fluorescence emission spectra of cells were taken using the gaseous argon laser (excited at 488 nm). The emission was collected from 510 to 695 nm using a 3 nm step. Acceptor photobleaching was carried out also using the Zeiss LSM 780 laser scanning confocal microscope. Images of AF488 and AF647 were obtained first. Then the cell was placed under 633 nm laser light at 100% intensity to bleach the AF647 until the intensity dropped to 5% of the original. Between every two scans, each pixel was exposed to laser irradiation for 1.5 μs each time and 50 times repeatedly. Then, the average increase in the donor (AF488) fluorescence in the cell after AF647 photobleaching was measured. The efficiency of energy transfer (E) was given by the following equation: E = 1 – FDA/FD, where FD and FDA are the relative fluorescence intensities of the donor in the absence (FD) or presence (FDA) of the acceptor.
3
Live Cell Fluorescence Microscopy Imaging
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Fluorescence microscopy using live cells was performed using an inverted confocal laser scanning microscope (LSM780; Carl Zeiss, Germany). For 3D and time-lapse images, HeLa cells expressing RFP-B23 were excited at a wavelength of 561 nm, and the emission signal band was detected at 570–630 nm. The interval for time-lapse imaging was 3 min, and z-stack images were taken at 0.5 µm intervals using a 5× magnification objective lens (C-Apochromat, 63×/1.2NA). All live cell measurements were performed at 37 °C in 5% CO2 culture conditions. Fluorescence images were processed using software (Zen 2012 SP5; Carl Zeiss, Germany) installed on an LSM780 confocal microscope system for 3D reconstruction or intensity profile analyses.
4
HOTAIRM1 Regulation of DNA Damage Response
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U2OS cells were seeded in a chambered coverglass (Thermo Scientific) and transiently transfected with pcDNA-HOTAIRM1-6× MS2 (full-length, Δ5′, ΔE2 or Δ3′) and pcDNA-MCP-GFP or GapmeRs. Laser microirradiation was performed 48 h post-transfection using a laser-scanning confocal microscope (LSM 780, Carl Zeiss) and a 405 nm laser diode. After laser microirradiation, cells were fixed with 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 in PBS. Immunofluorescence was performed by sequential incubation with primary and secondary antibodies. Nuclei were counterstained in Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Samples were visualized using a laser-scanning confocal microscope (LSM 780, Carl Zeiss) coupled with an image analysis system.
For live cell imaging, U2OS cells that stably expressed GFP-fused Ku70, Ku80 or MDC1 (33 (link)) were transiently transfected with pLKO.1-shHOTAIRM1. Laser microirradiation and time lapse imaging were carried out in a SP5 X inverted confocal microscope (Leica Microsystems) using laser diodes at 405 nm and 488 nm, respectively.
For live cell imaging, U2OS cells that stably expressed GFP-fused Ku70, Ku80 or MDC1 (33 (link)) were transiently transfected with pLKO.1-shHOTAIRM1. Laser microirradiation and time lapse imaging were carried out in a SP5 X inverted confocal microscope (Leica Microsystems) using laser diodes at 405 nm and 488 nm, respectively.
5
Multiscale Imaging of Drosophila Embryogenesis
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Cuticles were imaged on a Zeiss LSM780 with a 25× objective using a 550 nm laser. The resulting stacks were processed in Amira 5.32 (FEI). Four steps of blind deconvolution and Gaussian smoothing were applied. Intensity levels were set to range from 5-200. In situ stainings of germ bands were imaged on a Zeiss Axioplan2, 10× objective, using ImagePro 6.2 software (Media Cybernetics). Blastoderm in situ stainings were recorded as 8-bit mono for enhanced signal to overcome quenching and epifluorescence of the yolk. EdU-stained embryos were imaged on a Zeiss LSM780 with a 10× objective using 405 and 480 nm lasers. The resulting stacks were loaded in Amira 5.32 and 3D rendered with the voltex module. All images were assembled in Photoshop CS2 (Adobe). All figures were imported into Inkscape (http://www.inkscape.org/ ) for labeling and formatting.
6
Visualizing Filopodia Formation in Neurospheres
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For visualization of filopodia formation, neurospheres were incubated in Alexa Fluor 488-conjugated phalloidin reagents at room temperature for 30 min. Then, samples were covered with the ProLong Gold Antifade Reagent with DAPI, and the images were taken by a confocal microscope (LSM780, Carl Zeiss, Weimar, Germany) and investigated using Zen 2011 software (LSM780, Carl Zeiss, Weimar, Germany).
7
Visualization of AOM Consortia
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Organisms in AOM consortia were identified using catalyzed reporter deposition fluorescence in situ hybridization (CARD‐FISH) with clade specific probes following standard procedures (Pernthaler and Amann, 2004 ) and visualized with an epifluorescence or confocal laser scanning microscope (LSM 780; Zeiss; Oberkochen, Germany). The detailed employed CARD‐FISH protocol can be found in the Supporting Information. Oligonucleotide probe specifications are listed in Supporting Information Table S9. To visualize the autofluorescence of cofactor F420, AOM aggregates were sampled from the enrichments, transferred to microscopy slides and immediately examined using a confocal laser scanning microscope (LSM 780; Zeiss; Oberkochen, Germany) with an excitation light of 390–420 nm and a ≥ 463 nm emission filter.
8
Quantifying Nuclear Localization of NF-κB
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Images were acquired using a Zeiss LSM 780 mounted on a Zeiss axio observer inverted microscope. The 405 nm, 488 nm, 561 nm and 633 nm lasers were used, having been set with a unique and high-contrast color for each channel. The images were captured using the Zeiss ZEN 2011 software package associated with the LSM 780. Within ZEN 2011, the detectors were set at standard available ranges for specified dyes with little modification; pixel resolution was set at 1024x1024, at 8-bit color depth with a line scan and an averaging setting of 4. Prior to acquisitions, saturations and zeros were set using the range indicator.
For imaging, two tracks were created with two lasers each, in such a way that there would be no interference between laser channels. The 405 nm and 561 nm lasers were added into track 1 and the 488 nm and 633 nm lasers were added into track 2. The 405 nm laser was used to capture DAPI, the 488 nm was used to illuminate the AlexaFluor 488-Phalloidin, the 561 nm was aimed at exciting the Cy3-conjugated secondary and the 633 nm was used to target the AlexaFluor 647-conjugated secondary. We examined the total amount of NF-κB in the nucleus–as measured by the nuclear co-localization of NF-κB and DAPI. This was calculated as the nuclear fraction, i.e., the fraction of total cell fluorescence intensity coming from the nucleus.
For imaging, two tracks were created with two lasers each, in such a way that there would be no interference between laser channels. The 405 nm and 561 nm lasers were added into track 1 and the 488 nm and 633 nm lasers were added into track 2. The 405 nm laser was used to capture DAPI, the 488 nm was used to illuminate the AlexaFluor 488-Phalloidin, the 561 nm was aimed at exciting the Cy3-conjugated secondary and the 633 nm was used to target the AlexaFluor 647-conjugated secondary. We examined the total amount of NF-κB in the nucleus–as measured by the nuclear co-localization of NF-κB and DAPI. This was calculated as the nuclear fraction, i.e., the fraction of total cell fluorescence intensity coming from the nucleus.
9
Photoswitchable reporter in live MEFs
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MEFs transfected with the photoswitchable reporter pKFERQ-PS-CFP2 were cultured in µ-Slide Angiogenesis (IBIDI, 81506, Planegg, Germany). The photoconversion was carried out with a confocal laser scanning microscope (Lsm 780, Carl Zeiss, Oberkochen, Germany) by applying a 405 nm diode laser, 30 mW, 63× objective. The photoswitching time did not exceed 5 s in plane mode. The photoconversion did not affect the viability of the cells (data not shown). After treatment pictures of the cells were acquired using confocal laser scanning microscope (Lsm 780, Carl Zeiss) equipped with 63×1.4NA oil objective lens, a diode laser 405 nm and an argon laser, multiline, 458 nm, 488 nm, 514 nm, at 37C and 5% CO2. Pictures were analyzed with the ZEN 2010 software (Zeiss) and the quantification was done with Fiji software (Schindelin et al., 2012 (link)). Both the photoswitching and the imaging were performed in Phenol Red-free media.
10
Histological Evaluation of Cartilage and Chondrocyte Analysis
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After fixing in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and embedding in paraffin, 5 μm sagittal sections of cartilage specimens were cut and stained with hematoxylin and eosin (H&E), safranin O solution, and toluidine blue (Sigma-Aldrich) for histological examination. After washing with PBS (Servicebio, Wuhan, China), chondrocytes grown on slides were fixed with 4% paraformaldehyde for 30 min. Cells were then cocultured with a fluorescence in situ hybridization probe targeting tRF-5009A. After washing with PBS, cells were stained with DAPI. Images were captured using a confocal microscope (LSM780; Carl Zeiss, Germany). Immunofluorescence was performed 48 h after transfection, as previously reported [30 (link)]. The primary antibody used was against mTOR (1 : 100; Cell Signaling Technology, Danvers, MA, United States), while the conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, United States) was a goat anti-rabbit IgG. Images were obtained using a confocal laser microscope (LSM 780, Zeiss) at different magnifications.
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