D452-2 cells expressing the transporters were grown in oMM-Ura plus 2% glucose media to mid-exponential phase at 30°C. The cultures were centrifuged, spotted onto glass slides and examined on a Leica DM 5000B Epi-fluorescence microscope at 100x DIC (Leica, Germany). Transporters were visualized using the L5 filter cube; images were captured using the Leica DFC 490 camera and analyzed with the accompanying microscope software.
Dfc490 camera
Manufactured by Leica camera
Sourced in Germany
The DFC490 is a high-resolution digital camera designed for laboratory and scientific imaging applications. It features a 12.5-megapixel CMOS sensor and supports a variety of video and still image capture modes. The camera is capable of delivering detailed and accurate visual data for a range of microscopy and imaging tasks.
Automatically generated - may contain errors
Lab products found in correlation
X gluc, by Merck Group (2 mentions)
Qwin software, by Leica camera (2 mentions)
Leica dmlb microscope, by Leica camera (2 mentions)
Leica dmirb inverted microscope, by Leica camera (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Prolong diamond antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal microscope, by Nikon (1 mentions)
Plan apochromat 63x 1.4 na, by Nikon (1 mentions)
Mz6 stereomicroscope, by Leica camera (1 mentions)
Dm5000b microscope, by Leica camera (1 mentions)
Las af, by Leica camera (1 mentions)
6 well plate, by BD (1 mentions)
Pronase, by Merck Group (1 mentions)
Mz8 stereoscope, by Leica camera (1 mentions)
Anti digoxigenin ap antibody, by Roche (1 mentions)
T7 rna polymerase, by Roche (1 mentions)
Nbt bcip developing solution, by Roche (1 mentions)
Pgem t, by Promega (1 mentions)
Diaplan, by Leica camera (1 mentions)
19 protocols using dfc490 camera
1
Microscopic Visualization of Membrane Transporters
2
Immunofluorescence of Drosophila Tissues
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Indirect flight muscle was dissected and fixed in 4% formaldehyde (Agar scientific; R1926) in PBS for 30 minutes, washed twice with PBS, and mounted on slides in Prolong Diamond Antifade mounting medium (Thermo Fisher Scientific; RRID:SCR_015961 ). Larval epidermal cells were prepared as previously described (Lee et al., 2018 (link)). Larvae were dissected in PBS and fixed in 4% formaldehyde, for 30 min, permeabilized in 0.3% Triton X-100 for 30 min, and blocked with 0.3% Triton X-100 plus 1% bovine serum albumin in PBS for 1 h at room temperature. Tissues were incubated with anti-ATP5A antibody diluted in 0.3% Triton X-100 plus 1% bovine serum albumin in PBS overnight at 4°C, rinsed three times 10 min with 0.3% Triton X-100 in PBS, and incubated with the appropriate fluorescent secondary antibodies for 2 h at room temperature. The tissues were washed twice in PBS and mounted on slides using Prolong Diamond Antifade mounting medium (Thermo Fisher Scientific). Fluorescence imaging was conducted with a Zeiss LSM 880 confocal microscope/Nikon Plan-Apochromat 63x/1.4 NA oil immersion objective. For adult eyes, images were acquired using a Leica DFC490 camera mounted on a Leica MZ6 stereomicroscope set at maximum zoom.
3
Microscopic Imaging and Analysis Protocol
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Live squeeze preparations and H.E. stained sections were observed using a Leica DM 5000 B microscope and photographs were taken with a Leica DFC 490 camera. Confocal stacks were generated using a Leica TCS SP5 II confocal microscope and processed with the open‐source program Fiji v. 1.52j (Schindelin et al., 2012 ). Depth‐color‐coded images were done by using the look‐up tables “Ice” and “Spectrum” included in Fiji. Picture editing and drawings were done with the open‐source programs GIMP up to v. 2.10 (http://www.gimp.org ) and Inkscape v. 0.92 (https://inkscape.org ).
4
Zebrafish Embryo Drug Screening
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Embryos were collected in E3 at 28 °C then sorted and synchronised at shield stage (6hpf). One hour prior to drug addition, embryos were dechorionated using Pronase (Sigma) and washed extensively in E3 without methylene blue. Methylene blue was omitted from all subsequent E3 during drug treatment due to a reaction with NCI-65828 resulting in precipitation. Up to 50 embryos were transferred to 1.5% agarose coated 6-well plates (BD Falcon) in 2 ml of E3. Drugs were prepared as 100x stocks in DMSO; 8 mM NCI-65828 and either 2 mM or 3 mM terrein. At either 10 or 18hpf drugs were added to a final concentration of 80 µM NCI-65828, 20 µM terrein and 30 µM terrein with 0.1% DMSO used as control. 0.2 mM 1-phenyl 2-thiourea (PTU) was added at 24hpf. Observations were made using a Leica MZ8 stereoscope with images acquired with LAS AF (Leica) and videos recorded through VLC (videolan.org) through a Leica DFC490 camera. Embryos were washed twice with E3 before fixation in 4% paraformaldehyde (PFA, Sigma) pH7.4 overnight at 4 °C. After fixation embryos were washed twice with PBS and dehydrated through 30%, 50%, 70% to 80% methanol, each for 1 h.
5
Whole Mount In Situ Hybridization of Pax3 in E9.5 Embryos
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Whole mount in situ hybridization on E9.5 control embryos and embryos treated with BaP was performed according to the procedures described by Yun et al. [49 (link)]. Pax3 probe was cloned by real-time PCR into pGEM-T (Promega) and used to generate digoxygenin-labeled cRNA probes by reverse transcription using T7 RNA polymerase (Roche). For detection, anti-digoxigenin-AP antibody (1:2000, Roche) in 1% sheep serum was used and incubated overnight. Color detection was carried out using NBT/BCIP developing solution (Roche) in NTMT. After color development, embryos were imaged with a DFC490 camera (Leica), and then embedded. Sections of 40 μm thickness were obtained using a vibratome.
6
Microscopic Imaging of Biological Specimens
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Live and slightly squeezed animals as well as histological sections were observed and documented using a Leitz Diaplan or a Leica DM 5000 B light microscope equipped with a Motic Moticam 1080 camera or a Leica DFC 490 camera, respectively. Confocal stacks of fluorescently stained animals were made with a Leica TCS SP5 II confocal microscope. Images were analyzed and processed with the open‐source software Fiji v. 1.52j (Schindelin et al., 2012 ). The look‐up tables “Ice”, “ICA 3”, “physics” and “mpl plasma”, included in Fiji, were used to create depth‐color‐coded images. Schemes were drawn with the open‐source software Inkscape v. 0.92 (https://inkscape.org ) and picture editing was done using the open‐source software GIMP v. 2.10.8 (https://gimp.org ).
7
GUS Staining of Transgenic Roots
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The DR5:GUS insertion line grown as above was harvested and rinsed in distilled water for 30 s. After fixing in 90% (wt/wt) acetone in vacuo, the roots were rinsed with GUS staining buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 0.1% Triton X-100, 2 mM K4[Fe(CN)6], 10 mM EDTA) 3 times and then stained with 5 mL of GUS staining buffer containing 5 μg of X-gluc (Sigma-Aldrich, USA) for 2h, then they were immersed in the GUS staining buffer for observation. Images were photographed under a microscope (DM4000B equipped with a DFC490 camera, Leica, Germany).
8
Cytospin Microscopy Sample Preparation
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Cells were cytospun (Cytospin 3, 300 rpm for 5 minutes; Shandon, Runcorn, UK), air-dried, stained with Microscopy Hemacolor (Merck, Millipore, Billerica, Massachusetts, USA), visualized on a Leica DMLB microscope with DFC490 camera (Leica, Wetzlar, Germany) and processed using QWin Software (Leica).
9
Histological Preparation of Tissue Specimens
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Euthanized stages were fixed in Bouin fixative (picric acid plus formaldehyde plus acetic acid) for between 24 h and 1 wk. The specimens were decalcified using a decalcifying solution (0.5 M EDTA, pH 7.0) for 30 min, washed in 1× phosphate-buffered saline (PBS) three times (20 min each), dehydrated in ethanol, and followed by three xylene washes. Subsequent paraffin washes were conducted at 70 °C, and, finally, tissues of interest were embedded in paraffin/paraplast molds. Paraffin-embedded specimens were sectioned at 10 µm using a rotatory microtome and mounted onto Fisherbrand Suprafrost microscope slides. Both sagittal and transverse sections were left overnight on a slide-heater at 35 °C. The sections were stained as follows: Initially, deparaffinization was done in two xylene washes, sections were rehydrated using an ethanol series and stained with Mayer hematoxylin (2 min) to visualize nuclei, and the slides were kept under running water for 20 min. Next, eosin stain was used to visualize cytoplasm and extracellular matrix. Subsequently, dehydration was done in an ethanol series and, finally, in xylene. The slides were mounted in xylene-based DPX, sealed with a coverslip (no. 1 thickness), observed under a Zeiss Axio Imager 2 microscope, and photographed using a Leica DFC 490 camera.
10
Phenotypic Scoring of Zebrafish Larvae
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The zebrafish larvae were visually evaluated at 5 dpf using a fluorescence microscope (Fluorescence Stereo Microscope M205 FA, equipped with DFC490 camera, Leica, Mannheim, Germany). This study was non-blinded. Mortalities were noted as described above and surviving larvae were scored according to phenotypic abnormalities. Phenotypic scoring was based on the presence of abnormal pigmentation, presence of swelling of the region around the heart for pericardial oedema, presence of a dense red region in the head for cerebral haemorrhage, tail kinking for tail malformation, and angle deviation of the lower jaw compared to the larvae in negative control for jaw malformation [6 (link)]. Each larva was scored individually from (0) healthy, (1) mild, (2) moderate, and (3) severe malformations in every phenotype. Deceased larvae were excluded from the scoring system.
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