Microscopic images were acquired at 23°C with confocal microscopes (LSM 710/780; Carl Zeiss) using ZEN 2012 software and the following lenses: Plan-Apochromat 20×/0.8 NA M27, C-Apochromat 40×/1.20 NA W Korr M27, and Plan-Apochromat 63×/1.40 NA oil differential interference contrast (DIC) M27. Immersion medium Immersol W 2010 (ne = 1.3339) and Immersion oil Immersol 518 F (ne = 1.518) were used, respectively. For quantitative analysis, photon-counting mode was applied. Images were analyzed with Fiji v1.48 (Schindelin et al., 2012 (link)). 3D deconvolution was performed with Huygens Professional 14.06 Beta (Scientific Volume Imaging). Figure panels were finally assembled using CorelDRAW X4 (Corel Corporation).
Lsm710 780
Manufactured by Zeiss
Sourced in Germany
The LSM710/780 is a high-performance laser scanning microscope system developed by Zeiss. It is designed for advanced imaging applications in research and scientific laboratories. The system provides precise control of laser excitation and efficient light detection to enable high-resolution imaging and analysis of a wide range of samples.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ts2, by Nikon (2 mentions)
Coreldraw x4, by Corel (1 mentions)
880 laser scanning confocal microscope, by Zeiss (1 mentions)
Plan apochromat 63 1.40 oil lens, by Zeiss (1 mentions)
Can get signal immunostain solution, by Toyobo (1 mentions)
Fluorosave, by Merck Group (1 mentions)
αplan fluar 100 1.45 oil lens, by Zeiss (1 mentions)
Lsm 880 airyscan, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Diamino 2 phenylindole dapi, by Beyotime (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Vectashield mounting solution, by Vector Laboratories (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Hal 100 w, by Philips (1 mentions)
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Primary antibody dilution buffer, by Beyotime (1 mentions)
Rabbit anti cd31, by Abcam (1 mentions)
10 protocols using lsm710 780
1
Confocal Microscopy Imaging Protocol
2
Immunofluorescence Imaging of Cysts and Transwells
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Cysts and transwells were fixed with 4% methanol-free formaldehyde for 60 min at room temperature. Cysts were washed and permeabilised by three 20 min washes in wash buffer (1.3 M NaCl, 132 mM Na2HPO4, 35 mM NaH2PO4, 75 mM NaN3, 0.5% BSA, 2% Triton X-100, 0.4% Tween-20). Transwells were permeabilised in 0.3% Triton X-100 for 30 min and blocked in 5% BSA containing 15% FCS for 1 h. Next, cysts and transwells were incubated with primary antibodies diluted in 3% BSA in PBS overnight, followed by a 2 h secondary antibody incubation, and counterstained with the DNA dye Hoechst 33342 and Alexa Fluor 488/647 phalloidin. Confocal images were acquired on a Zeiss LSM 710, 780 or 880 confocal laser-scanning microscope. At least ten images were captured for each representative image depicted in the figures, and a minimum of three experiments was performed (n indicated in each figure).
3
Immunostaining of Adherent and Spheroid Cells
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Cells grown on cover glasses were fixed with 1% (wt/vol) PFA in the medium at RT for 10 min. In the case of sphere cultures, 2% (wt/vol) PFA was used for fixation. Cells were made permeable with 0.5% Triton X-100 in TBS for 20 min. The samples were blocked with 3% (wt/vol) BSA and 10% (vol/vol) goat serum in TBS containing 0.1% Triton X-100 (TBS-T) and incubated with primary antibodies in the Can Get Signal immunostain solution (TOYOBO) at RT for 2 h. After washes, the cells were incubated with fluorescence-labeled secondary antibodies for 1 h. After further washes, cells were incubated with fluorescence-labeled phalloidin (Molecular Probes) for 30 min and subsequently mounted using FluoroSave (EMD Millipore). Images of cells were obtained using a laser-scanning confocal microscope LSM710/780 (ZEISS) equipped with an αPlan-FLUAR 100×/1.45 oil lens or Plan-Apochromat 63×/1.40 oil lens (ZEISS) at RT. Z-stack images were taken at every 0.3 µm. For observation of the lateral views with higher resolution, images were obtained by the laser-scanning confocal microscope LSM880-Airyscan (ZEISS) equipped with an αPlan-FLUAR 100×/1.45 oil lens at RT. Z-stack images were taken at every 0.17 µm and subjected to Airyscan super-resolution mode processing. Images were processed using ZEN software (ZEISS) and Photoshop CS5 (Adobe Systems).
4
Cell-Laden Microfiber Imaging
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Optical images of cell-laden microfibers were obtained by an inverted microscope (ECLIPSE Ts2, Nikon, Tokyo, Japan). Cells seeded in microfibers were stained with LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA) to investigate the viability. Cytoskeleton and nuclei were stained with TRITC phalloidin and DAPI, respectively, according to the manufacturer’s instructions. Fluorescence photographs of cells-laden microfibers were taken by a fluorescence microscope (EVOS f1, Mill Creek, WA, USA). Fluorescence photographs of cross section of the cell-laden hollow microfibers were taken by a Laser Scanning Confocal Microscope (Zeiss LSM710/780, Oberkochen, Germany).
5
Immunohistochemical Analysis of Testis Sections
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IF experiments were performed as described by Wang et al. (2019) (link). After drying at room temperature for 15 min, the frozen testis sections of male L. crocea were permeabilized with 0.3% PBS with Triton X-100 (PBS-Triton X-100) for 20 min, followed by blocking with 5% bovine serum albumin in 0.1% PBS-Triton X-100 for 1.5 h and incubation with primary antibody overnight at 4°C. The frozen sections were washed with 0.1% PBS-Triton X-100 to remove the excess primary antibody and were incubated with the secondary antibody for 1 h. Excess secondary antibody was removed using 0.1% PBS-Triton X-100, and diamino-2-phenylindole (DAPI; Beyotime, China) was used to stain the nuclei for 5 min. Finally, the testis sections were mounted with Antifade Mounting Medium (Beyotime, China) and viewed using a confocal laser-scanning microscope (LSM710/780; Carl Zeiss, Oberkochen, Germany).
6
Immune-histology Analysis of Cultured Cells
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For immune-histology analysis, cells were fixed in 4% PFA for 20 min at room temperature. The cultured cell sample fixation time was between 11:00 and 15:00 for immunofluorescence analysis. Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 20 min. Blocking was performed with 10% FCS (Invitrogen) in PBS with 0.1% Triton X-100 for 1 h. Cells were incubated with primary antibodies diluted in blocking solution at room temperature for 2–3 h or overnight, washed twice in PBS, and reacted with secondary antibodies diluted in blocking solution for 45 min, counterstained with DAPI, and mounted with Vectashield mounting solution (Vector Laboratories). Primary antibodies were listed at the Table S2 were applied overnight at 4°C. Secondary antibodies were incubated for 2 h at room temperature. Confocal images were taken on standard fluorescence microscopes or Zeiss LSM710/780 confocal microscopes . All data for one experiment were acquired from cells cultured and processed in parallel on the same microscope with the exact same setting used.
7
Multimodal Confocal Imaging of Plant Cells
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Fluorescent images were obtained under a Zeiss LSM710/780 confocal microscope (Carl Zeiss Microscopy). Imaging was performed with a 40× and 63× oil-immersion lens in multitrack mode with line switching. cpYFP was excited at 488 (I488) and 405 nm (I405), and the emission signals were detected at 505–530 nm. AT1.03-nD/nA was excited at 458 nm at 7.5%, 10%, and 15% of maximal power for protoplasts, seedlings, and chloroplasts, respectively, and the emission signals were detected at 470–507 nm (Em470–507, mseCFP image) and 526–545 nm (Em526–545, FRET image). AT1.03-nD/nA was also excited at 515 nm (0.18% of maximal power for all samples) to directly excite Venus and detected at 526–545 nm (cp173Venus image). Chlorophyll fluorescence was detected at 629–700 nm. SYTOX orange nucleic acid stain was excited at 543 nm (1% of maximal power), and the emission signals were detected at 565–604 nm. White light illumination was achieved using a halogen lamp (HAL 100 W; Philips) attached to the confocal system.
8
Neural Crest Tissue Explant Analysis
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MOs and RNAs were injected with RNAs of GFP-Rab35 (50 pg) and Clip-E-cadherin (25 pg) into the D.1.2 blastomere at the 16-cell stage. GFP-positive neural crest tissues were dissected from stage 20 embryos and incubated with Clip-tag substrate (Non-cell-permeable, Clip-Surface™ 547) for 1 h at room temperature in 1× MBS. After brief washing with MDM, the neural crest tissue explants were placed on fibronectin-coated plates in MDM and incubated for 30 min to allow the explants to attach to the plates. Time-lapse images were taken using confocal microscopy (Zeiss LSM710/780).
9
Single-molecule RNA FISH of Platr4 in ESCs
10
Evaluating RUVEC-BMV Cell Viability
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Optical images of RUVEC-BMVs were obtained by an inverted microscope (ECLIPSE Ts2, Nikon, Tokyo, Japan) at 1, 3 and 5 days. After culturing for 5 days, the cells in RUVEC-BMVs were performed s live/dead staining to investigate the cell viability through laser scanning confocal microscope (Zeiss LSM710/780, Oberkochen,
Germany).
In addition, RUVECs were cultured in the BMVs (3D) and on the hydrogel at 3 and 14 days, the CD31 expression was tested via immunofluorescence. Briefly, RUVECs were fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.3% Triton X-100 in PBS for 10 min. Non-specific binding was blocked by 4% bovine serum albumin at room temperature for 1 h. After incubation with primary antibodies (rabbit anti-CD31, 1:400, Abcam, Cambridge, UK) in primary antibody dilution buffer (Beyotime, Shanghai, China) at 4 °C overnight, appropriate Cy3-labeled second antibodies (Beyotime, Shanghai, China) were incubated and DAPI for 1 hour at room temperature. Images were acquired using a fluorescence microscope.
Germany).
In addition, RUVECs were cultured in the BMVs (3D) and on the hydrogel at 3 and 14 days, the CD31 expression was tested via immunofluorescence. Briefly, RUVECs were fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.3% Triton X-100 in PBS for 10 min. Non-specific binding was blocked by 4% bovine serum albumin at room temperature for 1 h. After incubation with primary antibodies (rabbit anti-CD31, 1:400, Abcam, Cambridge, UK) in primary antibody dilution buffer (Beyotime, Shanghai, China) at 4 °C overnight, appropriate Cy3-labeled second antibodies (Beyotime, Shanghai, China) were incubated and DAPI for 1 hour at room temperature. Images were acquired using a fluorescence microscope.
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