The Motic® SMZ-168 Stereo Zoom Dissecting Microscope was used for worm phenotypic analysis. Homozygous mutants were selected from heterozygous worms with balancers using Olympus SZX2-ILLB microscope. All imaging for statistical analysis was done using Leica DM6 B upright microscope with the Leica DFC7000 T camera and LAS X Software, with the exception of RAD-51 foci, which was done using Zeiss LSM800 confocal microscope with Airyscan. All whole germline single layer images were captured using a Zeiss LSM800 confocal microscope with 10× objective at a scale bar of 50 μm, except for tm7141;nse-1::gfp with a remarkably small germline in which 40× objective with oil immersion was used at a scale bar of 10 μm. All other images were captured using a Zeiss confocal microscope LSM 800 with Airyscan at 63× objective with oil immersion (scale bar = 10 μm). Oocytes were scored for statistical analysis using Leica DM6 B at 100× objective with oil immersion. RAD-51 foci scoring was carried out with Z-stack images using 488 filter of Zeiss confocal microscope LSM 800 with Airyscan at 63X objective with oil immersion (scale bar = 10 μm). The RAD-51 fluorescence intensity for each gonad was normalized to the gonad rachis background of each gonad set using the wild-type.
Lsm 800
Manufactured by Zeiss
Sourced in Germany, United States, United Kingdom, Japan, Switzerland, France, China, Italy, Brazil, Australia, Canada
The LSM 800 is a laser scanning microscope designed for high-resolution imaging. It features a confocal optical system that allows for the acquisition of optical sections, enabling the visualization of 3D structures within a sample.
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Lab products found in correlation
Lsm 700, by Zeiss (148 mentions)
Lsm 880, by Zeiss (125 mentions)
Zen software, by Zeiss (63 mentions)
Hoechst 33342, by Thermo Fisher Scientific (60 mentions)
Lsm 780, by Zeiss (60 mentions)
Lsm 710, by Zeiss (58 mentions)
Airyscan, by Zeiss (57 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (50 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (49 mentions)
Triton x 100, by Merck Group (41 mentions)
Zen blue software, by Zeiss (40 mentions)
Axio observer z1, by Zeiss (34 mentions)
Lsm 510, by Zeiss (33 mentions)
Zen 2, by Zeiss (30 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (29 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (28 mentions)
Plan apochromat, by Zeiss (21 mentions)
Paraformaldehyde (pfa), by Merck Group (20 mentions)
Bovine serum albumin (bsa), by Merck Group (19 mentions)
Vectashield, by Vector Laboratories (17 mentions)
2 673 protocols using lsm 800
1
Worm Phenotypic Analysis Using Microscopy
2
Live Imaging of Dorsal and Erk in S2R+ Cells
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S2R+ cells were plated on 35 mm glass-bottom dish at 80% confluence and transfected as mentioned. Cells were imaged 24 h after transfection using the Zeiss LSM800 confocal microscope with 63× oil immersion lens. Cells transfected with either Dorsal or Erk were used as negative controls for respective experiments. For live embryo imaging, embryos were processed as for Lightsheet microscopy and mounted in 1% low melting point agarose on glass bottomed petri dishes. Imaging was done on a Zeiss LSM 800 (Carl Zeiss, Germany) using the following settings: 1% laser power for 488 nm; 5% laser power for 561 nm. Images were processed using the ZEN 2014 SP1 software (Carl Zeiss, Germany).
3
3D Spheroid Imaging and Analysis
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Anti-β1 integrin AlexaFluor488 (clone P5D2) was stained at a concentration of 1:200 (v/v), phalloidin AlexaFluor 647 was stained at a concentration of 1:1000 (v/v), and DAPI at a concentration of 1:1000 (v/v). Samples were stained overnight in the dark at 4 °C and were subsequently rinsed three times for 10 min with 1× PBS. Samples were overlaid with 100 μL of 1× PBS and then imaged (while still in 3D) on the Zeiss LSM800 using a 20× objective. Z-stacks were taken at 10 μm intervals as high as the working distance of the objective would allow. Samples were then overlaid with gelatin and PBS, frozen and cryosectioned (DISC-3D Protocol Part 2). Every slice (typically, ≈ 50) of the spheroid was imaged on the Zeiss LSM800 using the 20× objective. Each of these slices was then re-stained using the same dyes and concentrations listed above. Approximately 20 μL of the staining solution was placed atop each individual slice of the cryosectioned spheroid, and the samples were allowed to sit overnight at 4 °C in a humidified and sealed chamber (so as to reduce evaporation of the staining solution). The samples were rinsed the next morning three times in 1× PBS for 10 min, dried completely, and every slice was again imaged as described above.
4
Confocal Microscopy Imaging Workflow
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We observed samples under a confocal microscope (Zeiss LSM 800, Carl Zeiss, Jena, Germany) with a 20 × (NA 0.80) or 63 × (NA 1.4) objective lens (LSM 800, Carl Zeiss). Images were captured using a charge-coupled device camera (Axio-Cam MRm, Carl Zeiss), transferred to a computer, and analyzed using ZEN software (Carl Zeiss).
5
PDE2A Expression Analysis in Mouse Cortex
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At the time of euthanasia, one hemisphere per mouse was post fixed in a solution of 4% paraformaldehyde at 4 °C for 48 h, dehydrated in graded ethanol solutions, cleared in xylene, and embedded in paraffin. Serial sections (6 µm thick) were cut onto positively charged glass slides and rehydrated in ethanol solutions of decreasing concentrations. Slides were boiled in citrate buffer (pH 6.0) for 7 min for antigen retrieval, transferred to a Sudan Black solution for 15 min to prevent autofluorescence and blocked for 1 h with UltraCruz Blocking Reagent (Santa Cruz: sc-516214). Fluorescent staining was performed with the antibodies for PDE2A (FabGennix: PD2A-101AP) during the overnight incubation. On the next day, secondary antibodies AlexaFluor555 (Thermo Fisher Scientific: A31572) were applied. Slides were mounted with ProLong Gold Antifade 4',6-diamidino-2-phenylindole (DAPI) Mount. Imaging was performed using a confocal microscope (LSM 800 Zeiss) at 20× magnification. Quantification of the fluorescent images was performed using the LSM 800 Zeiss and the number of cells per selected region of interest (ROI) in the cortex was measured.
6
Quantitative Analysis of Demyelination, Axonal Loss, and Monocyte Infiltration
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To assess demyelination, axonal loss, and monocyte infiltration, images were acquired at 20× on the Zeiss LSM800. At least three images per section were acquired in two sections per animal and at least three animals were analyzed for each condition. Images were taken as a Z-stack and the maximum projection was acquired using the Zeiss Zen software. Areas were calculated on ImageJ. Fluoromyelin and NFH+ areas were delineated manually. To calculate CD45+ area, images were thresholded and the area calculated using the “analyze particles” function. Regions of interest (ROIs) were defined as CD45+ areas, and Iba1+ area was calculated within CD45+ areas using a threshold. Total Iba1+CD45+ area for each image was calculated as the sum of Iba1+ area for all CD45+ areas or ROIs in each image. ROIs were limited to the lesion area. To assess DNA methylation levels, images were acquired at 63× on the Zeiss LSM800. 3 images per section were taken and 2 sections per animal and 3 animals per condition were assessed. Intensity was defined as mean gray value and calculated on Image J. 5 representative nuclei of Iba1+ cells were selected manually in each image and 5mc and DAPI intensity were calculated. For statistical analysis, Student's t-test was performed on Graphpad Prism v7.0 and p values <.05 were considered significant.
7
Muscle Regeneration Immunofluorescence Protocol
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For immunofluorescence staining of Pax7, EdU, Myog and eMyhc, the Gas muscles were harvested at day 3. Immunofluorescence staining on frozen muscle sections was performed in accordance with previous reports21 (link), and images were visualized using a confocal laser scanning microscope (Zeiss, LSM800, Germany). The following dilutions were used for each antibody: Pax7 (Developmental Studies Hybridoma Bank; USA; 1:20), eMyhc (Developmental Studies Hybridoma Bank, USA; BF-G6; 1:100), Myog (Santa Cruz Biotechnology, USA; sc-12732; 1:20). To detect the EdU incorporation, the sections were performed using the Life Technologies Click-iT Kit according to the manufacturer’s instructions, and images were photographed using a confocal laser scanning microscope (LSM800; Zeiss). For H&E staining, the Gas muscles were harvested at day 0, 3, 7, and 15 after CTX injection. H&E of muscle sections was performed according to previous reported methods21 (link),45 (link), and the cross-section area of individual myofibers was visualized using Olympus DP80 upright Metallurgical Microscope (Olympus Corporation, Japan) and qualified using ImageJ software.
8
Imaging Techniques for Primary Microglia
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9
Imaging of DEV and Oil Droplets
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10
Measuring Intracellular ROS in HCEC-B4G12 Cells
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The production of intracellular ROS in HCEC-B4G12 cells was detected by H2DCF-DA staining as described previously [30 (link)]. Briefly, H2DCF-DA (10 μM) was used to treat HCEC-B4G12 cells for 30 min in a cell culture incubator. To remove the unbound dye, the cells were washed three times with PBS and then exposed to Na-Mt and C-H-Na-Mt of various concentrations (3.13–50 μg/mL) with a phenol red-free medium. Subsequently, the cells were incubated continuously and the oxidation of H2DCF-DA was later detected by CLSM (Zeiss LSM 800, Germany) at 2, 6, 12, and 24 h. In vivo, the levels of corneal ROS were measured via DHE staining of 8-μm-thick corneal cryosections. Briefly, cryosections were fixed with 4% paraformaldehyde at room temperature for 60 min. Then, the sections in 10 μM DHE were incubated for 30 min in the dark, after which DAPI was added to counterstain and seal each after rinsing with PBS for corneas. Three sections of each rat were analyzed and photographed using CLSM (Zeiss LSM 800, Germany).
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