Mosquitoes were screened for the presence of DsRed marker using a Leica M165 FC microscope equipped with a DsRed filter. Images were captured with a Leica DFC365 FX camera mounted on a Leica M165 FC microscope.
M165fc microscope
Manufactured by Leica
Sourced in Germany, Israel
The M165FC microscope is a high-performance stereo microscope developed by Leica. It features a high-contrast, bright optical system and a flexible stand for a wide range of applications. The M165FC provides continuous magnification from 7.8x to 160x, allowing for detailed observation and inspection of specimens.
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Lab products found in correlation
Alizarin red, by Merck Group (5 mentions)
Dfc500, by Leica (5 mentions)
Dfc3000g camera, by Leica (4 mentions)
Dfc425c, by Leica (3 mentions)
Camera dfc 3000g, by Leica (3 mentions)
El6000, by Leica (2 mentions)
Nile red, by Merck Group (2 mentions)
Wormlab software, by MBF Biosciences (2 mentions)
Las software, by Leica (2 mentions)
Ds fi2 digital camera, by Nikon (2 mentions)
Dfc310 fx camera, by Leica (2 mentions)
Axio imager a2 microscope, by Zeiss (2 mentions)
Alcian blue, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Az100, by Nikon (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dfc425 camera, by Leica (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
122 protocols using m165fc microscope
1
Visualizing DsRed in Mosquitoes
2
Zebrafish Embryonic Manipulation and Analysis
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DMH1 (Sigma-Aldrich, St. Louis, MI, United States, D8946) used in experiments was reconstituted at stock concentrations (10 mM) in DMSO solvent. Embryos were dechorionated by hand using forceps and dechorionated embryos were transferred to 6-well plates. Embryo media with appropriate drug concentration or vehicle control was added to each well. For DMH1 treatments, embryos were treated from 28 hpf to 76 hpf. Embryos were incubated at 28.5°C for the duration of the drug treatment. For phenotypic analysis, embryos were mounted in 2% methylcellulose and documented using Leica M165FC microscope and Leica MC 170 HD camera. P values were calculated using unpaired t-test with Welch’s correction (GraphPad Prism7).
The sequence of alk3a, alk3b, alk6a, and alk6b morpholinos were used as previously described (Little and Mullins, 2009 (link); Neumann et al., 2011 (link)) (Supplementary Table 1 ). Each morpholino was injected into 1-cell stage wild-type zebrafish embryos. The morpholino doses used are 1, 2, 4, 8, 16, and 32 ng. Embryos were collected at 52 and 76 hpf and mounted in 2% methylcellulose and documented using Leica M165FC microscope and Leica MC 170 HD camera.
The sequence of alk3a, alk3b, alk6a, and alk6b morpholinos were used as previously described (Little and Mullins, 2009 (link); Neumann et al., 2011 (link)) (
3
Visualizing and Quantifying P. infestans Colonization
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P. infestans 88069td colonization of Arabidopsis was visualized using a Leica M165FC microscope with DFC425 camera and EL6000 light source (Leica Microsystems, Milton Keynes, UK) and a DSR filter (excitation wavelength of 510–560 nm and emission wavelength of 590–650 nm). P. infestans growth is represented by red fluorescence. Leaves that were inoculated with P. infestans on the abaxial surface may show no fluorescence from the adaxial surface due to lack of pathogen colonization (e.g. Col-0 plants).
P. infestans colonization of Arabidopsis was quantified using qRT-PCR. Leaf discs (10 mm diameter) were punched out of Arabidopsis leaves inoculated with P. infestans and DNA extracted with DNeasy plant mini kit (Qiagen, Hilden, Germany). Four discs were used per replicate for water-sprayed plants, and three discs per replicate for Albugo-sprayed plants. DNA was diluted to 5 ng/μL and 5 μL used per qRT-PCR reaction. qRT-PCR was conducted as described below, using primers for At3g21215 and PiO8-3-3 (Additional file4 ) to compare the amount of P. infestans DNA present.
P. infestans NL12226 sporulation on Col-0 and cyp79b2/b3 Arabidopsis was quantified by infecting leaves from 4-week-old plants (as described above), then checking for the presence of P. infestans spores between 3 and 5 dpi by placing droplets of water on the leaf surface and examining them under a light microscope.
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P. infestans 88069td colonization of Arabidopsis was visualized using a Leica M165FC microscope with DFC425 camera and EL6000 light source (Leica Microsystems, Milton Keynes, UK) and a DSR filter (excitation wavelength of 510–560 nm and emission wavelength of 590–650 nm). P. infestans growth is represented by red fluorescence. Leaves that were inoculated with P. infestans on the abaxial surface may show no fluorescence from the adaxial surface due to lack of pathogen colonization (e.g. Col-0 plants).
P. infestans colonization of Arabidopsis was quantified using qRT-PCR. Leaf discs (10 mm diameter) were punched out of Arabidopsis leaves inoculated with P. infestans and DNA extracted with DNeasy plant mini kit (Qiagen, Hilden, Germany). Four discs were used per replicate for water-sprayed plants, and three discs per replicate for Albugo-sprayed plants. DNA was diluted to 5 ng/μL and 5 μL used per qRT-PCR reaction. qRT-PCR was conducted as described below, using primers for At3g21215 and PiO8-3-3 (Additional file
P. infestans NL12226 sporulation on Col-0 and cyp79b2/b3 Arabidopsis was quantified by infecting leaves from 4-week-old plants (as described above), then checking for the presence of P. infestans spores between 3 and 5 dpi by placing droplets of water on the leaf surface and examining them under a light microscope.
4
Visualizing Silkworm Fat Body Organs
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Fat body organs from silkworm larvae were collected at the indicated times and then fixed in 4% paraformaldehyde for 30 min at room temperature. After fixation, the samples were washed three times with phosphate-buffered saline (PBS). The washed fat body samples were incubated with Nile Red (1:1000, Sigma-Aldrich, St. Louis, MO, USA), DAPI (1:1000, Life Technologies, Carlsbad, CA, USA) and phalloidin (1:500, Life Technologies, Eugene, OR, USA). Fluorescence signals were captured by confocal microscopy (Olympus, Tokyo, Japan) at the excitation wavelengths of 488 and 594 nm. White light signals were captured using an M165FC microscope (Leica Microsystems, Wetzlar, Germany).
5
Quantifying Worm Motility under Stressors
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Synchronized worms were transferred on 35 mm Ø NGM agar plates with HT115 at 20 °C and recorded on day 4 of adulthood. In total 4 conditions were compared: control N2 with M9 buffer, N2 treated with mtPQ 0.1 μM, control mcu-1 mutants with M9 buffer and mcu-1 mutants treated with DTT (Sigma Aldrich, #1610611) 10 mM. For the treatment, 1 μL of M9 buffer, mtPQ (Cayman, #CAY-188085) or DTT solution were directly pipetted on the individual worms under the microscope and incubated for 20 min before recording the worms. Three plates of 10 worms each were recorded for 45 s per condition. Plates were recorded using a Leica M165 FC microscope with a DFC7000 T 2.8 MP camera (Leica Microsystems) connected to a computer. The recorded videos were used to calculate the distance covered by the worms according to the organism's center of gravity using the Parallel Worm Tracker for MATLAB [52 ]. Based on this, the average speeds of the worms per plate and condition were calculated with ObjectAnalyzer and further analyzed with Excel and Graphpad Prism 7.02 [53 ].
6
Vero Cell Viability Assay
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Vero cells were plated at 5 × 104 cells/well in 12-well plates and incubated overnight at 37 °C with 5% CO2, then infected with 0.1 MOI of Z1 and Z7 (G11) and incubated for 1 h at 37 °C. The medium was then replaced with 1 ml of DMEM supplemented with 10% FBS and 1% P/S and incubated for 3 days. On D3 p.i., the cells were stained using LIVE/DEAD Cell Imaging Kit (488/570, ThermoFisher Scientific) according to the user’s manual. The images were taken using a Leica M165 FC microscope (Leica Microsystems).
7
Visualizing Adipocytes in Zebrafish Larvae
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Wild type zebrafish was handled and maintained according to the standard protocol (Kimmel et al., 1995 (link)). Larvae obtained from daily crosses were raised and fed regular diet starting at 6 days post fertilization (dpf). Treatment of either DMSO or CA (at 10 μM final concentration) was done for 8 days starting from 10 to 17 dpf (n = 15 per group). To visualize adipocytes, larvae were treated with Nile red (Sigma Chemicals, St. Louis, MO, USA) and imaged with a Leica M165FC microscope (Leica Microsystems, Wetzlar, Germany). The intensity of fluorescent signals was quantified by ImageJ 1.47v (National Institute of Health, Bethesda, MD, USA).
8
Embryo Development in Timed Mating Mice
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For timed mating experiments, 3- to 4-month-old stud males were housed individually for 1 to 2 weeks prior to mating. Females that are 8 to 15 weeks old were group housed for 10 to 14 days prior to mating and were exposed to soiled bedding from a male’s cage 2 days before mating. One to 2 female(s) in estrus were added into each stud male’s cage at 9:00 PM. Females were examined for vaginal plugs and were separated from male mice at 9:00 AM the next morning. The morning when a vaginal plug was found was set as embryonic day 0.5 (E0.5). At 9:00 AM in the morning of E10.5 or other embryonic stages as indicated, pregnant females were killed. Embryos were isolated, and images of yolk sacs and embryo proper were captured on Leica M165FC microscope (Leica Microsystems GmbH, Germany).
9
Multi-modal Microscopy Imaging Protocol
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Brightfield images were captured using a Leica M165FC microscope with a DFC450 C color camera (Leica Microsystems, Wetzlar, Germany). Fluorescent imaging was performed using a Zeiss Axio Zoom.V16 microscope with an Axiocam 305 color camera (Carl Zeiss Microscopy, Jena, Germany). Zstack recordings were reconstructed to a single in-focus image using the complex wavelet-based Extended Depth of Field plugin in ImageJ (FIJI distribution, version 1.52n, NIH) (Forster, Van De Ville, Berent, Sage & Unser, 2004 (link), Schindelin, Arganda-Carreras, Frise, Kaynig, Longair et al., 2012) (link). For scanning electron microscopy (SEM), the sample was mounted on an aluminium base and sputtered with platinum using the JEOL JFC 1300 Auto Fine Coater (Jeol Ltd., Tokyo, Japan). The sample was examined with a JEOL JSM 5600 LV scanning electron microscope (Jeol ltd.). Multiple images were stitched together using the ImageJ plugin developed by (Preibisch, Saalfeld & Tomancak, 2009) (link).
10
Confocal Imaging of Retinal and Brain Tissues
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All images of retinal and brain sections were acquired by a Zeiss LSM880 inverted confocal microscope. Retina explants were imaged by Leica M165 FC microscope. Images in Figures 2 and 4 were maximum projections of 5 to 10 μm tissues and were quantified by Fiji software. Images in Figures 3 and 5 were projections of 30 μm tissues.
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