In situ hybridization of transcripts for cloned genes was performed using protocols established for chromogenic detection in the cnidarian Nematostella vectensis [56 (link)]. A detailed protocol is presented in Additional file 3 . Hybridization was performed at 62°C for 48 h with DIG-UTP-labeled probes at a concentration of 1 ng/μL. Detection of hybridized probes was performed by staining with NBT and BCIP, after labeling with alkaline phosphatase-conjugated anti-DIG antibody. At least 20 embryos were processed per stage for each gene, and development of staining was checked by a stereomicroscope prior to completion of the in situ protocol and mounting for imaging. In all cases, staining was highly consistent within stages. Embryos were cleared and mounted in 80% glycerol, and imaging was performed on a Zeiss AxioSkop microscope equipped with Plan-Apochromat 20×/08 N.A. objective and differential interference contrast optics (Carl Zeiss, Jena, Germany). Images were acquired with a Zeiss AxioCam HRc digital camera and Zeiss AxioVision v4.8 software (Carl Zeiss, Jena, Germany).
Plan apochromat 20 0.8 na objective
Manufactured by Zeiss
Sourced in Germany
The Plan Apochromat 20×/0.8 NA objective is a high-performance microscope objective designed by Zeiss. It features a magnification of 20× and a numerical aperture of 0.8. The objective is optimized for flat, aberration-free image field.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (3 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Axioskop microscope, by Zeiss (1 mentions)
Axiovision v4, by Zeiss (1 mentions)
Axiocam hrc digital camera, by Zeiss (1 mentions)
Imt 2, by Olympus (1 mentions)
Schneider s insect medium, by Merck Group (1 mentions)
Apotome 2, by Zeiss (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Lsm 5 live, by Zeiss (1 mentions)
Mvx10, by Olympus (1 mentions)
Axio observer z1 7, by Zeiss (1 mentions)
Lsm 900 confocal, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Duo92102, by Merck Group (1 mentions)
Lsm 710 microscope, by Zeiss (1 mentions)
Bx60 upright microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
14 protocols using plan apochromat 20 0.8 na objective
1
In Situ Hybridization of Nematostella Transcripts
2
Fluorescence Microscope Imaging System
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The imaging system consists of an inverted fluorescence microscope (IMT-2, Olympus), mounted with a Plan-Apochromat 20× /0.8NA objective (Carl Zeiss) and a fluorescence filter cube; a Royal-Blue LED (Luxeonstar) served for the fluorophore excitation. A CCD (GX1920, Allied Vision Technologies) was mounted via zoom and 0.1× c-mount adaptors (Vario-Orthomate 543513 and 543431, Leitz), sampling at 70 Hz, 968 × 728 px, covering 810 × 610 μm laterally and the full depth of the tube. The camera control was based on a modification of the Motmot Python camera interface package35 (link), expanded with a home-made plug-in, to allow real-time image analysis in the RAM23 (link), recording only the time-lapse positions of the tracers to the hard drive.
3
Immediate Imaging of Vesicle Structures
4
Zebrafish Imaging Techniques Protocol
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Routine bright-field and fluorescence imaging of zebrafish used an Olympus MVX10 stereo dissecting microscope (Olympus, Tokyo, Japan) with MV PLAPO 1× and 2×C objectives, fitted with an Olympus DP72 camera and Cellsense standard software, version 1.11.
Confocal intravital microscopy used a Zeiss LSM 5 Live with a Plan-Apochromat 20×, 0.8 NA objective (Zeiss, Oberkochen, Germany). ZEN software (2012, black edition, 64-bit) was used for acquisition, and images were 16-bit 512 × 512 pixels. Z-depth ranged from 35–130 μm (72 ± 23 μm) and was composed of 20–40 slices (31 ± 4). Time intervals between z-stacks were set as zero to perform continuous acquisition (z-stack acquisition took 33.24 ± 9.50 s). Excitatory laser wavelengths were 405 nm for calcofluor, 489 nm for EGFP, and 561 nm for mCherry. Emission detection used a BP495-555 filter for calcofluor and EGFP and an LP575 filter for mCherry. Excitation/emission conditions for light yellow particles were the same as for calcofluor.
Details of microscopes, cameras, and acquisition software used for other experiments are provided with their respective methods.
Confocal intravital microscopy used a Zeiss LSM 5 Live with a Plan-Apochromat 20×, 0.8 NA objective (Zeiss, Oberkochen, Germany). ZEN software (2012, black edition, 64-bit) was used for acquisition, and images were 16-bit 512 × 512 pixels. Z-depth ranged from 35–130 μm (72 ± 23 μm) and was composed of 20–40 slices (31 ± 4). Time intervals between z-stacks were set as zero to perform continuous acquisition (z-stack acquisition took 33.24 ± 9.50 s). Excitatory laser wavelengths were 405 nm for calcofluor, 489 nm for EGFP, and 561 nm for mCherry. Emission detection used a BP495-555 filter for calcofluor and EGFP and an LP575 filter for mCherry. Excitation/emission conditions for light yellow particles were the same as for calcofluor.
Details of microscopes, cameras, and acquisition software used for other experiments are provided with their respective methods.
5
Confocal Microscopy Analysis of Coated Wood
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One beech wood sample spray coated with EOL-TC was investigated using a Zeiss LSM 900 confocal laser scanning microscope equipped (Oberkochen, Germany)on Axio Observer Z1/7 inverted stand, with diode lasers with 405 nm, 488 nm, 561 nm, and 640 nm wavelengths, and using a plan-apochromat 20×/0.8NA objective (Carl Zeiss S.p.A., Milan, Italy). The measurements were conducted in a measurement area of 319.45 × 319.45 µm using fluorescence excitation for the specific illumination of lignin (553 nm) and carbohydrate (401 nm) moieties on the coated wood sample surface. The emission wavelengths were 568 nm for lignin and 422 nm for cellulose.
6
Multimodal Imaging of Cell Lines
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Fluorescence measurements were conducted using a confocal laser scanning microscope CLSM 780 meta (Carl Zeiss, Jena, Germany) for measurements of the THP-1 cells and a CLSM 980 (Carl Zeiss, Jena, Germany) for characterizing the A549 and CaCo-2 cells. Images were acquired using a Plan Apochromat 20×/0.8 NA objective (Carl Zeiss, Jena, Germany) for overview images of four-to-six fields of view (FOV), each about 350 µm × 350 µm in size, whereas a Plan Apochromat 40×/0.95 NA objective (Carl Zeiss, Jena, Germany) was used for additional detailed images. Dragon Green polystyrene beads were excited with 488 nm and detected in the range of 490–571 nm and I555 phalloidin was excited using a 561 nm laser and detected in the range of 562–615 nm, while DAPI was excited with 405 nm and detected in a range of 409–493 nm, additionally detecting bright field to reveal cellular localization. To avoid channel crosstalk, all channels were recorded sequentially. Images were acquired in different focal planes (z-stack) at a distance of 1 µm and covering the entire height of the cell layer.
7
Quantifying Nuclear Membrane Ruptures
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Mice expressing the Nuc-tdTomato transgene were perfused with 3% PFA in PBS and tissues postfixed in the same solution at 4°C. Frozen tissue sections (10 μm–thick) were stained with DAPI and mounted in Antifade (Invitrogen). Images were captured on a Zeiss LSM 800 laser-scanning confocal microscope controlled by Zen Blue 2.3 software using a Plan-Apochromat 20×/0.8 NA objective. Maximum image projections were generated from scans of the tdTomato and DAPI channels, combined with the autofluorescence signal in the GFP channel. The latter was used to identify the elastic fibers in the media layer. A NM rupture was defined as the appearance of tdTomato fluorescence outside of a nucleus, as judged by DAPI staining. If tdTomato fluorescence was present between 2 adjacent nuclei, this was counted as a single NM rupture. NM ruptures were counted and expressed relative to the total number of nuclei examined.
8
Proximity Ligation Assay in HeLa Cells
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HeLa cells were fixed in 3.7% formaldehyde for 10 min at room temperature and permeabilized by 0.3% Triton X-100 solution in PBS. After washing three times with PBS, a standard PLA procedure was performed as recommended by the manufacturer (DUO92102; Sigma–Aldrich). In brief, cells were treated with blocking solution for 1 h. One of the primary antibodies (anti-YB1, anti–Ago-1, anti–Ago-2, Smad-1, or Lamina-1) was diluted 1:200 and added to the cells for an overnight incubation at 4 °C. Diluted (1:5) PLA probes were added to the cells after two consecutive 5-min washes (wash buffer A) and incubated for 1 h at 37 °C. Following an additional 5-min wash with buffer A, diluted (1:5) ligation stock was added to the samples and the incubation was extended for an additional 30 min. Cells were washed twice for 2 min with buffer A. The amplification step was performed by applying a mix of 1:5 diluted amplification stock and 1:80 diluted polymerase to the cells and incubating for 100 min at 37 °C. Cells were washed with buffer B several times and mounted for imaging using Duolink In Situ Mounting Medium with DAPI. After ∼15 min, Z-section fluorescent images were acquired with a Zeiss LSM880 confocal microscope using a Plan-Apochromat 20×/0.8 N.A. objective.
9
Visualizing Root Epidermal Cell Organization
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For imaging of cell file organization in root epidermis, living roots were stained with 4 μM of the styryl dye FM4-64 in liquid ½ MS medium in order to delineate cell borders and documented with a Zeiss LSM710 microscope. Plants were observed with Plan-Apochromat 20×/0.8 NA objective and data sets were analyzed with Zeiss Zen 2014 software (Blue Version).
10
Correlative In Vivo and Ex Vivo Imaging
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Multiple cold-cut biopsies were harvested from each rat. Brain tissue slices (0.5 cm2) containing several ROIs were cut parallel to the surface. After the in vivo imaging, tissue was imaged ex vivo with a Zeiss 710 inverted laser scanning confocal microscope. For comparison to the CLE, we equipped the Zeiss 710 with a Plan Apochromat 20 × 0.8 NA objective, and set the confocal aperture for 1 Airy unit. Areas imaged using the confocal microscope and CLE were marked with tissue ink so that locations could be precisely correlated and validated with conventional histology. The tissue was placed in a cassette for standard formalin fixation and paraffin embedding. Histologic assessment was performed using standard light microscopic evaluation of 5- μm thick H and E–stained sections with an Olympus BX60 upright microscope. Careful labeling and grouping of acquired confocal images and specific biopsy samples ensured correct correlation.
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