Samples were diluted in fixative as required to obtain an evenly spread preparation, and 8 μl of sample dropped onto a slide and allowed to air dry. Slides were stained with 16 μl VectorShield with DAPI (Vector Labs) under a 22 × 50 mm cover slip and imaged using an Olympus UPFLN100XOI2 100× oil immersion plan semiapochromat objective (NA 1.30) on an Olympus BX-61 epifluorescence microscope equipped with a Hamamatsu Orca-ER C4742-80 cooled CCD camera and appropriate filters. Images were captured using Smart-Capture 3 (Digital Scientific, UK). To validate the reproducibility of the software, sample images were also gathered on three other microscopes: (1) an Olympus BX61 with a Hamamatsu C10600 orca r² camera, (2) an Olympus BX61 with a Hamamatsu Orca-03G camera (both (1) and (2) using an Olympus UPFLN100 × 100× oil immersion plan semiapochromat objective [NA 1.30]), and (3) a Nikon Microphot-SA epifluorescence microscope using a Nikon 100× oil immersion plan apochromat objective (NA 1.40) with a Photometrics Metachrome II CH250 cooled CCD camera.
Orca er c4742 80
Manufactured by Hamamatsu Photonics
Sourced in Germany, Japan
The ORCA-ER C4742-80 is a high-performance charge-coupled device (CCD) camera developed by Hamamatsu Photonics. It features a back-thinned, back-illuminated CCD image sensor with a resolution of 1344 x 1024 pixels. The camera offers high quantum efficiency, low read noise, and fast frame rates, making it suitable for a variety of scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1 fluorescence microscope, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Microphot sa, by Nikon (1 mentions)
Vectorshield with dapi, by Vector Laboratories (1 mentions)
Myogenin myog, by Cell Signaling Technology (1 mentions)
Hoechst 33342 nuclear dye, by Thermo Fisher Scientific (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Dm6000, by Leica (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Metamorph software package, by Molecular Devices (1 mentions)
Simplepci software, by Hamamatsu Photonics (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Dmrxa2, by Leica (1 mentions)
Np 40, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bx61wi, by Olympus (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Softworx software, by Cytiva (1 mentions)
Iplab software, by BD (1 mentions)
Hoechst 33342, by Nacalai Tesque (1 mentions)
14 protocols using orca er c4742 80
1
Fluorescence Microscopy Imaging Protocol
2
Nanofluidic Protein Concentration Analysis
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The nanofluidic device was inserted into
a plastic holder (SI Figure S6 ). The protein
sample and buffer solutions were continuously flowed into the nanofluidic
device. The details are described in theSI . The fluorescence signal from each postconcentration channel was
detected by a CCD camera (ORCA-ER C4742-80, Hamamatsu) with a motorized
stage (P-H101P1F, Prior Scientific) at a regular interval (10 min).
The signal from each postconcentration channel was analyzed by ImageJ
software.6 (link) It was normalized by a background
signal and was averaged over 2 h (n = 12).
a plastic holder (
sample and buffer solutions were continuously flowed into the nanofluidic
device. The details are described in the
detected by a CCD camera (ORCA-ER C4742-80, Hamamatsu) with a motorized
stage (P-H101P1F, Prior Scientific) at a regular interval (10 min).
The signal from each postconcentration channel was analyzed by ImageJ
software.6 (link) It was normalized by a background
signal and was averaged over 2 h (n = 12).
3
Immunofluorescence Quantification of Myogenic Differentiation
4
Myotube Contractility Analysis on Elastomers
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Zeiss Axio Observer Z1 (LSM 510; Zeiss, Oberkochen, Germany) equipped with a digital camera (ORCA-ER C4742-80; Hamamatsu, Bridgewater, NJ) was used to record the beating of mouse myotubes after 10 days of differentiation of myoblasts on P(GTP-co-HDT), P(GTP-co-BDBMP), P(GTP-co-TMPTMP), and P(GTP-co-ETTMP) elastomers. The spontaneous beating frequency was obtained by counting the number of beatings per minute. The density of beating myotubes on the surface was counted for 10 different fields of view (area = 0.55 mm2 per field of view).
5
Immunocytochemical analysis of airway epithelial cells
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Airway epithelial cells (both 16HBE’s and BEAS2B’s) were grown on the collagen coated 15 mm coverslips (Chemglass Life Science, Vineland, NJ) and subjected to immunocytochemistry staining. At Days 1, 2 and 4 after DA exposure, cells were fixed for 10 min using 4% paraformaldehyde (PFA) and then permeabilized with 0.1% Triton-X-100 in PBS for 15 min. Fixed cells were then blocked with 2% bovine serum antigen (BSA) in phosphate-buffered solution (PBS) for 1 h and then incubated with the primary antibody of integrin beta 4 (ITGβ4, CD104; 1:250, Abcam, Cambridge, MA), Keratin 5 (KRT5; 1:250, Invitrogen, Waltham, MA), or integrin alpha 6 (ITGα6; 1:250, Biolegend, San Diego, CA) overnight at 4 °C. Cells were washed, incubated with the fluorescent secondary antibody (1:500–1000, Invitrogen, Waltham, MA) for 1 h, and mounted with DAPI Fluoromount medium (Southern Biotech, Birmingham, AL). Images were acquired using a fluorescence phase contrast microscope (Leica DM6000, Wetzlar, Germany) equipped with a digital camera (Hamamatsu orca-ER C4742-80, Japan). All image processing was performed using ImageJ program (NIH, Bethesda, Maryland).
6
Immunofluorescence Staining of Cultured Cells
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Immunofluorescence staining was performed as described previously [27 (link)]. Briefly cells at the indicated conditions were fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature; permeabilized with 0.1% (v/v) Triton‐X100 in PBS (10 min, room temperature); and incubated in blocking buffer (5% [v/v] goat serum in PBS/0.1% [v/v] Triton‐X100) at room temperature for 1 h. The cells were then incubated with primary antibodies (Table S1 ) overnight at 4 °C followed by three washes with PBS. Subsequently, samples were incubated with a secondary antibody (Table S1 ) at room temperature for 1 h. Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific) was used to stain for F‐actin as per manufacturer's instructions. Cell nuclei were counterstained with Hoechst 33342 (10 mg·mL−1; 1 : 200 dilution in PBS; 5 min at room temperature; Thermo Fisher Scientific). Stained cells were visualized with a Zeiss AxioVision imaging system (RRID:SCR_002677, Observer Z1 LSM 510 microscope; Zeiss, Oberkochen, Germany) equipped with a digital camera (ORCA‐ER C4742‐80; Hamamatsu, Bridgewater, NJ, USA) and fluorescent images were quantitatively analyzed using the NIH image j software.
7
Calcium-Induced Interphase Transition
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Calcium chloride was added to a final concentration of 0.8 mM in metaphase-arrested extracts to send them into interphase. Reactions were performed at 25°C for 60 min, and 2 µl of the reaction was mixed with 1 µl of 0.025% octadecyl rhodamine in buffer (10 mM Hepes, pH 7.4, 250 mM sucrose, 50 mM KCl, and 2.5 mM MgCl2). Images were acquired with a 60× oil objective lens (NA 1.42) on a microscope (BX61; Olympus) equipped with a charge-coupled device camera (ORCA-ER; C4742-80; Hamamatsu Photonics) at ∼20°C. Images were acquired and analyzed using the MetaMorph software package (Molecular Devices), and three-way junctions were counted to assess network formation.
8
Immunofluorescence Microscopy Protocol
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Cells cultured on glass coverslips were fixed either in methanol for 2 min at 4°C or in 4% paraformaldehyde for 10 min, followed by 0.1% Triton X-100 for 8 min at room temperature, depending on the performance of the primary antibodies used (Supplemental Table S2). Secondary antibodies conjugated to fluorescent dyes (Alexa Fluor 488, 555, or 647 nm; Life Technologies) were used to identify target molecules. Microscopy was performed using an epifluorescence microscope (DMRXA2; Leica, Wetzlar, Germany) equipped with 63×/1.32 numerical aperture (NA) and 100×/1.40 NA oil immersion objectives with apochromatic aberration and flat-field corrections, narrow-bandpass filters, and a digital camera (ORCA-ER C4742-80; Hamamatsu Photonics, Naka-ku, Hamamatsu, Japan). Images were captured using Simple PCI software (Hamamatsu Photonics).
9
VEGF Regulation of HUVEC Adhesion
10
Immunostaining of Small-Cell Lung Cancer Cells
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Small-cell lung cancer cells (NCI H345 and NCI H82) were seeded (104) onto glass coverslips and allowed to recover for 24 h at 37°C. The cells were treated with a paraformaldehyde fixative buffer, permeabilized with 0.05% NP40 (Sigma-Aldrich Chemical Co.) and non-specific staining blocked with a gelatin buffer. They were then incubated with MAG-1 antibody (or MOPC antibody control) diluted in PBS containing 0.1% BSA, washed five times with buffer, and then incubated with Alexa Fluor 488 goat anti-mouse secondary antibody (Molecular Probes) for 1 h, following manufacturer recommendations. Finally, cells were washed (×5) with 1% BSA in PBS, the nuclei of the cells sometimes stained using DAPI, cells post-fixed with 4% paraformaldehyde and mounted in Vectashield (Vector Laboratories) on glass slides. Slides were visualized with an Olympus BX61WI fluorescence confocal microscope employing a Hamamatsu Orca-ER C4742-80 camera.
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