Mouse LNs were isolated 72 h post-intradermal injection of model vaccines. LNs were fixed with 4% paraformaldehyde (Thermo Scientific, Rockford, IL) and incubated in 30% sucrose solution (Merck, Germany) overnight. Ten-μm thickness of frozen sections were cut by a cryostat (Leica, Germany). Images were acquired on a 4-channel NIR fluorescence microscope (TE2000U, Nikon, Japan) and CCD camera (C4742–80-12AG, Hamamatsu Photonics, Japan). All the images were binarized by means of iterative selection method to define areas positive for fluorescence signals. To analyze the spatial relationship between fluorescent cells and their locations in the LN, areas within 15 μm of the edge of LNs were defined as subcapsular sinus (SCS) and the others were considered as cortex.[34 (link)] The portion of areas positive for fluorescence was measured and calculated for each ROI using ImageJ.
C4742 80 12ag
Manufactured by Hamamatsu Photonics
Sourced in Japan
The C4742-80-12AG is a digital CCD camera designed for scientific and industrial applications. It features a high-resolution 1344 x 1024 pixel CCD sensor and a 12-bit analog-to-digital converter. The camera is capable of capturing images at a maximum frame rate of 15 frames per second. It is designed for use with Hamamatsu's DCAM interface and is compatible with a variety of image processing software.
Automatically generated - may contain errors
Lab products found in correlation
Ix71 microscope, by Hamamatsu Photonics (2 mentions)
Sc 2367, by Santa Cruz Biotechnology (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Sucrose solution, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Te2000 u, by Nikon (1 mentions)
El6000, by Leica (1 mentions)
Premiere pro, by Adobe (1 mentions)
M165 fc, by Leica (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Hcimage acquisition software, by Hamamatsu Photonics (1 mentions)
Fura 2am, by Nacalai Tesque (1 mentions)
Ix71 inverted microscope, by Hamamatsu Photonics (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Proscan stage, by Prior Scientific (1 mentions)
Ix71 fluorescent microscope, by Olympus (1 mentions)
Nis elements software program, by Nikon (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Bz 9000, by Keyence (1 mentions)
13 protocols using c4742 80 12ag
1
Visualizing Lymph Node Responses to Vaccines
2
Visualizing α-Tubulin in Microtubules
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MTs samples were labeled to visualize α-tubulin. The antibody raised from rabbit against amino acids 149–448 of human α-tubulin was obtained from Santa Cruz Biotechnology Inc (H-300, sc-5546) and used at 1:500 dilutions as previously reported19 (link). The secondary antibody used for tubulin staining was bovine anti-rabbit IgG-R (sc-2367, Santa Cruz Biotechnology Inc, CA) used at a 1/1000 dilution. Samples were viewed under an inverted Olympus IX71 microscope connected to a digital CCD camera C4742-80-12AG (Hamamatsu Photonics KK, Bridgewater, NJ). Images were collected with the IPLab Spectrum (Scanalytics, Viena, VA) acquisition and analysis software, running on a Dell-NEC personal computer.
3
Stereoscopic Imaging and Calcium Dynamics
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Stereoscopic images and videos were taken using a Leica M165 FC fluorescence stereo microscope, a Leica EL6000 lightsource, the Leica filter set ET GFP LP (Leica Microsystems, Germany), and a cooled CCD camera (Hamamatsu C4742-80-12AG, Hamamatsu Photonics, Herrsching, Germany). For GCaMP6f. calcium imaging, an excitation wavelength of 475 nm and an exposure time of 50 ms at 2× binning was used. Fluorescence videos were taken as a sequence of uncompressed tiff files in a range where the fluorescence intensities of samples and brightness values of captured images were linear. Ether was applied by adding 2 ml ether to a glass container next to the sample in a gas-tight setup creating an ether atmosphere of ~ 15%. 10× time laps for Movies S3 , S8 , S9 and S10 was achieved using Windows Movie Maker (Microsoft), Side by side views were produced in Premiere Pro (Adobe) and compression to MPEG2 was done in VLC media player (VideoLan). To calculate the propagation velocity of the [Ca2+]cyt increase a sample rate of 25 ms was chosen. Hairs and traps were background subtracted and the mean velocity was calculated over known distances. For quantitative analysis of areas with increased [Ca2+]cyt after stimulation two ROIs (regions of interest) with the same pixel numbers were defined in the indicated tissues using ImageJ. Data points were fitted using Igor Pro 8.
4
Blastocyst Permeabilization and Nuclear Staining
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Blastocysts were permeabilised in 0.1% Triton-100X/PBS during two minutes at room temperature and then rinsed twice in phosphate buffered saline (PBS). Finally, embryos were mounted on a SuperFrost microscope slide using Vectashield-DAPI as mounting medium and nuclear staining. Total cell count was effectuated on a Nikon Eclipse Ti 90× microscope, along with a Hamamatsu digital camera (C4742-80-12AG) and a fluorescent filter corresponding to the excitation 350 nm and 470 nm of emission for DAPI.
5
Immunochemical Labeling of Honeybee Brain MTs
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Honeybee brain MTs were immunochemically labeled with an anti-α-tubulin antibody raised in rabbit against amino acids 149–448 of human α-tubulin (H-300, sc-5546, Santa Cruz Biotechnology Inc) that was used at 1:100 dilution. The secondary antibody used for tubulin staining was a FITC-tagged bovine anti-rabbit IgG-R (sc-2367, Santa Cruz Biotechnology Inc, CA) used at a 1/100 dilution. Samples were viewed under DIC and fluorescence microscopy with an inverted Olympus IX71 microscope connected to a digital CCD camera C4742-80-12AG (Hamamatsu Photonics KK, Bridgewater, NJ). Images were collected with the IPLab Spectrum (Scanalytics, Viena, VA) acquisition and analysis software, running on a Dell-NEC personal computer.
6
Microfluidic Deformability Analysis of RBCs
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The microfluidic device was designed with layout program and it consists of 500 μm × 500 μm inlet/outlet reservoirs and parallel capillary channels with triangular pillar arrays (Fig. 1a ). Deformability device was mounted to the microscope (Olympus IX51, Center Valley, PA) connected to a CCD camera (Hamamatsu Model C4742-80-12AG). Approximately 3 μl of blood samples were loaded to reservoir of the microfluidic device. The inlet reservoir was connected to a vertically held 60-ml syringe which was partially filled with with RPMI medium containing 20% FBS buffer solution. A laminar flow was induced by gravity11 (link)13 (link)26 (link). Images were automatically obtained by IPLab (Scanalytics, Rockville, MD) at 100 ms time interval and the post-imaging analysis was done using imageJ. Cell deformability is defined by the transit velocity of cells flowing in the microchannels. The velocity of individual RBCs was simulated with the following equation:
where displacement is the distance the cells moved; the traverse time is in seconds. Normalized velocity was calculated by dividing the velocity of individual RBCs from various experimental conditions by the control group RBC velocity on the same experimental day.
where displacement is the distance the cells moved; the traverse time is in seconds. Normalized velocity was calculated by dividing the velocity of individual RBCs from various experimental conditions by the control group RBC velocity on the same experimental day.
7
Fluorescent Live Embryo Imaging
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Live embryos were imaged using a fluorescent compound microscope (Nikon Eclipse 80i, Nikon, Melville, NY, USA) equipped with a digital camera (Hamamatsu C4742-80-12AG). Embryos were anesthetized with 0.2 mg·mL−1 tricaine and placed in a 3 cm petri dish containing 1% agarose with larva-sized grooves for immobilization. Histological sections were imaged with a Zeiss LSM 710 inverted confocal microscope. Photostability was measured before and after 5 min of bleaching with a 488 nm laser.
8
Calcium Imaging of DRG Neurons
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A total of 20 rats, three C57BL/6 mice, and three TRPA1 deficient mice were used for calcium imaging analysis. Twenty-four hours after dissection of the DRG neurons, they were loaded with 4 μM Fura-2AM (Nacalai Tesque, Kyoto, Japan) for 1 h at 37 °C. The coverslip was placed into the recording chamber, and a normal bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (HEPES), and 10 mM glucose at pH 7.4, adjusted with NaOH, was perfused. Depolarization of the DRG neurons was induced with 50 mM KCl in a normal bath solution. Ratiometric calcium imaging was performed using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742-80-12AG, Hamamatsu Photonics, Hamamatsu, Japan). Dual images (at 340 and 380 nm excitation) were collected every 1 s and analyzed using HCImage Acquisition software (Hamamatsu Photonics, Hamamatsu, Japan). A microscopic field containing 20–30 neurons was randomly selected under the 20X objective.
9
Immunofluorescent Detection of Primary Cilia
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Confluent cell monolayers exposed overnight to the various experimental conditions were rinsed twice with phosphate-buffered saline (PBS) and fixed for 10 min in a freshly prepared solution of paraformaldehyde (4%) and sucrose (2%). Cells were then washed three times with PBS, blocked for 30 min with BSA (1%) in PBS, and incubated for 60 min with an anti-acetylated-α-tubulin antibody (Santa Cruz Biotechnology) to identify primary cilia (Raychowdhury et al., 2005 (link)). Anti-mouse IgG FITC-coupled (Invitrogen) was used as secondary antibody. Cells were counter-stained with DAPI to locate cell nuclei and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Cells were viewed under an Olympus IX71 inverted microscope connected to a digital CCD camera C4742-80-12AG (Hamamatsu Photonics KK, Bridgewater, NJ). Images were collected and analyzed with the IPLab Spectrum acquisition and analysis software (Scanalytics, Viena, VA), running on a Dell-NEC personal computer.
10
Automated Fluorescence Microscopy Quantification
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