Immunostaining of imaginal discs was performed as previously described (Matakatsu and Blair, 2004 (link)), except that fixations were performed in PBS with 2% formaldehyde for 5 min at RT. The following primary antibodies were used: rat anti–Dachs N and C (1:20,000), guinea pig anti-App (1:20,000; Matakatsu and Blair, 2008 (link)), mouse anti–β-galactosidase (1:1,000; Developmental Studies Hybridoma Bank), rabbit anti–β-galactosidase (1:1,000; Cappel), mouse anti-V5 (1:1,000; Invitrogen), and mouse anti-CD2 (1:1,000; Serotec). Images were taken using an LSM 880 confocal microscope (ZEISS; using Zen 2.1 software) or an Apotome (ZEISS) with an ORCA-ER CCD digital camera (Hamamatsu Photonics). Plan Apochromat 40×/1.4 and Plan Apochromat 63×/1.4 oil-immersion objectives (ZEISS) were used for LSM880. The acquired images were processed with ImageJ and Photoshop (Adobe Systems). Apical z-stacks were processed to make maximal projections using ImageJ.
Orca er ccd digital camera
Manufactured by Hamamatsu Photonics
The Orca-ER CCD digital camera is a high-performance scientific imaging device manufactured by Hamamatsu Photonics. It features a charge-coupled device (CCD) sensor with a high quantum efficiency, low noise, and rapid readout capabilities. The Orca-ER is designed for demanding applications requiring high-quality, low-light imaging.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Apotome, by Zeiss (2 mentions)
Mouse anti β galactosidase, by Developmental Studies Hybridoma Bank (1 mentions)
Mouse anti cd2, by Bio-Rad (1 mentions)
Plan apochromat 63 1.4 oil, by Zeiss (1 mentions)
Mouse anti v5, by Thermo Fisher Scientific (1 mentions)
Imagej, by Adobe (1 mentions)
Nis elements software, by Nikon (1 mentions)
Autoquant, by Media Cybernetics (1 mentions)
Uplsapo 60 1.3 na silicone oil objective, by Olympus (1 mentions)
Cellsens software, by Olympus (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Openlab software, by PerkinElmer (1 mentions)
Ix70 inverted microscope, by Hamamatsu Photonics (1 mentions)
Anti nestin, by BD (1 mentions)
Glucose catalase, by Merck Group (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
Fluorescence microscope, by Hamamatsu Photonics (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Ab6556, by Abcam (1 mentions)
11 protocols using orca er ccd digital camera
1
Immunostaining of Drosophila Imaginal Discs
2
Time-lapse Analysis of Mitotic Progression
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Time-lapse microscopy was performed using a Nikon TE2000-E motorized inverted microscope equipped with a complete incubation system (Nikon). Time-lapse images of H1299 cells were taken with the Orca-ER CCD digital camera (Hamamatsu Photonics) at 5-min intervals for 48 h. Anaphase onset was defined as the time the cell starts to round up. All images were processed using NIS Elements software (Nikon).
3
Microscopic Imaging and Deconvolution
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Images were acquired using cellSens software (Olympus) on an IX83 microscope (Olympus) with a UPLSAPO 60× 1.3 NA silicone oil objective (Olympus) and an Orca-ER CCD digital camera (Hamamatsu Photonics). Deconvolution was done with Autoquant (Media Cybernetics) standard settings for each filter set using a blind adaptive point-spread function. Image analysis was done in Fiji (http://fiji.sc/ ).
4
Immunocytochemistry of Neural Markers
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Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min at room temperature. After rinsing with PBS, cells were incubated with blocking/permeabilizing solution, which consists of PBS with 10% donkey serum and 0.1% Triton X-100 for 1 hr at room temperature. Subsequently, cells were incubated with primary antibodies overnight at 4°C. Primary antibodies used in this study were anti-nestin (1:1000; BD Sciences, USA) and anti-beta-III tubulin (1:500; Covance). In order to visualize the primary antibodies, cells were incubated with fluorescent-labeled secondary antibodies (Alexa 488-Alexa 594-, or Alexa 647-labeled IgG; Life Technologies) for 1 hr at room temperature. Images of stained cells were captured using an Olympus IX-70 inverted microscope, equipped with an Orca-ER CCD digital camera (Hamamatsu) and OpenLab software (Improvision). ImageJ (Wayne Rasband National Institute of Health, USA) was used for image analysis. In addition, images were analyzed by CellProfiler (version 2.1.1; cellprofiler.org ) for measuring fluorescent intensity. Three independent fields from each time point were used with matching exposure.
5
Immunofluorescent Microscopy of Candida albicans
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This procedure was done as described in [13 (link)] with minor modification. C. albicans cells were grown overnight in YPD or YPM medium at 30°C. Mouse anti-β (1,3)-glucan antibody (Biosupplies Australia Pty Ltd., Australia) at a 1:800 dilution was used as the primary antibody, and a goat anti-mouse antibody conjugated to Cy3 (Jackson ImmunoResearch Inc., USA) at 1:300 dilution was used as secondary antibody. For imaging, Candida cells were resuspended in 100 μL of PBS and visualized with LEICA DM5500B epi-fluorescent microscope with Hamamatsu Orca-ER CCD digital camera (Model#C4742-80-12AG). The pictures were taken through Leica Application Suite AF (A dvanced F luorescence) software.
6
Measuring Microtubule Depolymerization Dynamics
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A 0.24 µM mixture of AlexaFluor-555-labeled and unlabeled MTs were prepared in 1xPM (100 mM PIPES, pH 7.0, MgSO4, 1 mM EGTA) with 1 mM MgATP, 1 mM NaGTP, 0.4 µM Taxol, and gloxy [0.1 µg/ml glucose oxidase (Sigma), 2 µg/ml glucose catalase (Sigma)]. For TIRF measurements, 7 µl of 0.24 µM polymerized tubulin mixture was placed between a glass slide (1 mm thickness, 75 × 25 mm2) and plasma-cleaned coverslip (150 μm thickness, 22 × 22 mm2) for all measurements. For measurements with motor, a final concentration of 0.4 µM Eg5-367 or 0.2 µM Eg5-513 was used. For measurements with allosteric Eg5 inhibitor, concentrations are detailed in the figures.
Time-lapse imaging of depolymerizing labeled MTs with or without motor was performed on an Olympus IX81 inverted microscope using a 60X 1.49 NA Plan Apochromat TIRFM oil objective at room temperature. Images were acquired with a Melles Griot HeNe 543 nm laser and a Hamamatsu ORCA-ER CCD digital camera. All hardware was controlled by Micromanager software operating on the ImageJ platform.
Time-lapse imaging of depolymerizing labeled MTs with or without motor was performed on an Olympus IX81 inverted microscope using a 60X 1.49 NA Plan Apochromat TIRFM oil objective at room temperature. Images were acquired with a Melles Griot HeNe 543 nm laser and a Hamamatsu ORCA-ER CCD digital camera. All hardware was controlled by Micromanager software operating on the ImageJ platform.
7
Bacterial Induction and Microscopic Imaging
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Bacteria were grown overnight at 30°C in AB medium containing antibiotic (50 μg/mL kanamycin). To induce expression of a fusion gene, 5 mL AB induction medium (AB Mes, pH 5.8, 50 μmol/L acetosyringone, and 50 μg/mL kanamycin) was inoculated with 0.35 mL of the overnight culture and grown for 16–18 h at room temperature (Mossey et al. 2010 (link)). Seven microliters of induced cells were placed on a polylysine-coated glass slide, and a cover glass was placed on top. After 10 min on bench top to allow bacteria to adhere, samples were viewed with a Leica fluorescence microscope equipped with a Hamamatsu ORCA-ER digital CCD camera. Images were recorded with Volocity software (PerkinElmer, Waltham, MA) and processed using Adobe Photoshop CS2 for presentation. For quantitative analysis, number of labeled cells was counted and their fluorescence patterns were analyzed.
8
In Vivo VSD Imaging of Somatosensory Cortex
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VSD imaging was performed as described previously2 (link). 1–2% isoflurane was used to anesthetize the animals. After being placed in a stereotaxic apparatus, heating pad was used to maintain animal’s body temperature throughout the length of anesthesia. Each animal’s scalp was disinfected with betadine followed by isopropyl alcohol. An incision was made. A round 5–8 mm craniotomy was performed over somatosensory cortex. After dura removal, 1 mg/ml Rh1691 in phosphate-buffered saline (PBS) (Optical Imaging) was applied directly to the brain for 1 hour. Then the cortex was washed with PBS to remove unbound dye. A 5–8 mm glass coverslip was attached to the surrounding skull using dental cement and Krazy glue.
Wide field VSD imaging was carried out using a 2X long working distance objective (NA = 0.14) on an upright microscope (BX51, Olympus). 120 W mercury arc lamp was used to excite the VSD with 585/20 nm light. Hamamatsu ORCA-ER digital CCD camera was used to collect images with 5 ms exposure every 50 ms. Image sequences were acquired in 50 s epochs, up to 10 epochs/condition.
Wide field VSD imaging was carried out using a 2X long working distance objective (NA = 0.14) on an upright microscope (BX51, Olympus). 120 W mercury arc lamp was used to excite the VSD with 585/20 nm light. Hamamatsu ORCA-ER digital CCD camera was used to collect images with 5 ms exposure every 50 ms. Image sequences were acquired in 50 s epochs, up to 10 epochs/condition.
9
Indirect Immunofluorescence Assay for Plasmodium
10
Automated High-Throughput Microscopy Imaging
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