Zebrafish embryos were incubated for 2.5 h in the dark in 2.5μg/ml neutral red solution (Sigma Aldrich, n2889) diluted in Danieau’s solution. Afterwards, embryos were washed 3 times 10 minutes with Danieau’s solution. Brightfield images of the head of the fish were taken using a Leica DFC300 FX Digital Color Camera.
Dfc300 fx digital color camera
Manufactured by Leica camera
Sourced in Germany
The DFC300 FX Digital Color Camera is a high-quality digital camera designed for laboratory and scientific applications. It features a large sensor that captures detailed images with accurate color reproduction. The camera is capable of capturing images at a resolution of up to 5.0 megapixels and can be integrated with various microscopes and imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
Sodium azide, by Merck Group (2 mentions)
Dm6000b microscope, by Leica camera (1 mentions)
Rabbit anti human irf5 antibody, by Cell Signaling Technology (1 mentions)
Hoechst, by ImmunoChemistry Technologies (1 mentions)
Cy3 labeled goat anti rabbit igg antibody, by Jackson ImmunoResearch (1 mentions)
Dm irb inverted fluorescence microscope, by Leica camera (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Mz16 fa fluorescence stereomicroscope, by Leica camera (1 mentions)
Tricaine ms 222, by Merck Group (1 mentions)
Nahco3, by Merck Group (1 mentions)
Spss software version 22, by IBM (1 mentions)
Dm4000, by Leica camera (1 mentions)
Facscalibur, by BD (1 mentions)
7 protocols using dfc300 fx digital color camera
1
Neutral Red Staining of Zebrafish Embryos
2
Neutrophil Morphological Identification
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After purification by magnetic bead selection, CD66b+CD10+/–-neutrophil populations were stained by Reich Giemsa staining. Leica DFC 300FX Digital Color Camera on a Leica DM 6,000 B microscope at a 40× magnification was used to take picture. Both a pathologist and a hematologist, who were blinded to this study and cell samples, were assigned to identify the mature and immature neutrophil within at least 200 cells per slide of CD66b+CD10+/–-LDN/NDN.
The criteria of neutrophil morphology are as following: (1) Promyelocytes are characterized by a slightly often eccentrically indented nucleus in a blue cytoplasm; (2) myelocytes are characterized by a round or oval nucleus in a light blue cytoplasm; (3) metamyelocytes are characterized by a kidney-shaped nucleus in a pink cytoplasm; (4) band cells are smaller with a characteristic horseshoe-shaped nucleus of uniform thickness; and (5) mature neutrophils have a segmented nucleus typically characterized by 2–5 lobes separated by narrow filamentous bridge as illustrated inFigures 3B , 4B .
The criteria of neutrophil morphology are as following: (1) Promyelocytes are characterized by a slightly often eccentrically indented nucleus in a blue cytoplasm; (2) myelocytes are characterized by a round or oval nucleus in a light blue cytoplasm; (3) metamyelocytes are characterized by a kidney-shaped nucleus in a pink cytoplasm; (4) band cells are smaller with a characteristic horseshoe-shaped nucleus of uniform thickness; and (5) mature neutrophils have a segmented nucleus typically characterized by 2–5 lobes separated by narrow filamentous bridge as illustrated in
3
Immunofluorescent Detection of IRF5 Translocation
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For analysis of IRF5 translocation, DCs or macrophages were stimulated as indicated in Maxisorp plates. After 2 h stimulation, cells were washed with PBS, fixed with 3.7% formaldehyde (Sigma-Aldrich) for 15 min at room temperature, washed in PBS and stored in PBS containing 0.5% bovine serum albumin (BSA; PAA) and 0.1% sodium azide (Merck) at 4°C. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and blocked for 30 min in PBS containing 0.5% BSA and 0.1% sodium azide. Cells were then stained with a rabbit-anti-human-IRF5 antibody (1:400) (Cell Signaling) or rabbit-anti-human NF-kB p65 antibody (1:100) (Cell Signaling) for 45 min at room temperature, washed with PBS and stained with a Cy3-labeled goat-anti-rabbit-IgG antibody (1:50) (Jackson ImmunoResearch). Cells were again washed with PBS and nuclei were stained using 1 μg/mL Hoechst (Immunochemistry Technologies) for 1 min at room temperature. Cells were imaged using a DM IRB inverted fluorescence microscope (Leica), combined with a DFC 300FX digital color camera (Leica).
4
Quantitative Analysis of Larval Zebrafish Morphology
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Fish were sedated with 200 μg/ml ethyl-3-aminobenzoate methanesulfonate (Tricaine/MS222; Sigma; A5040) in system water buffered to pH 7.0–7.5 with NaHCO3 (Sigma; S5761) and imaged on moist filter paper with an MZ16 FA fluorescence stereomicroscope (Leica, Wetzlar, Germany) and DFC 300 FX Digital Color Camera (Leica) at 1.4 MPixel resolution at 7.11× and 14× magnification (55 ). Images were stitched together in Pixelmator 3.5 Canyon software (Pixelmator Team, Vilnius, Lithuania). Total body length was measured using Fiji software, and differences in mean total body length between genotypes were tested for significance by two-sided Student’s t-test. The number of fish with dorsal tilting of the head was analyzed by the Fisher Exact test in SPSS software version 22 (IBM, Armonk, NY, USA).
5
Neutral Red Staining of Zebrafish Embryos
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Zebrafish embryos were incubated for 2.5 h in the dark in 2.5 µg ml−1 of neutral red solution (Sigma-Aldrich, n2889) diluted in Danieau’s solution. Afterwards, embryos were washed three times for 10 min with Danieau’s solution. Bright-field images of the head of the fish were taken using a Leica DFC300 FX Digital Color Camera.
6
Imaging Microscopy of Anesthetized Animals
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Animals were mounted on 2% agar pads containing 0.2% levamisole
as the anesthetic and imaged using a Leica DM4000 with a 20×
objective and a Leica DFC300 FX Digital Color Camera.
as the anesthetic and imaged using a Leica DM4000 with a 20×
objective and a Leica DFC300 FX Digital Color Camera.
7
Toxoplasma Infection in MEFs
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For all infections, MEFs were plated in either 12-or 24-wells and the following day, or when monolayers were at a confluency of $70%, infected with Toxoplasma at an MOI of 2. Two hpi, infected monolayers were washed twice with serum-free media to rinse off extracellular parasites and either incubated in cDMEM alone or supplemented with the following inhibitors or reagents from Sigma-Aldrich: syntheChol NS0, linoleic acid-oleic acid-albumin, atglistatin (as indicated), GW0742 (50mM), and etomoxir (200mM), pyrimethamine (40mM), and MitoTEMPO (20mM) At 24hpi, cells were rinsed with PBS, trypsinized and fixed in 2% paraformaldehyde in FACS buffer (3% FBS in PBS) for 10min. After a brief spin, cells were resuspended in FACS buffer and sorted on a FACScalibur (BD Biosciences) and analyzed for median FI (mFI) using CellQuest Pro, at least s000 infected cells counted per sample. Infected cells were identified by the red fluorescence of the transgenic Toxoplasma. If provided, brightfield and fluorescence images of live monolayers were captured at 24hpi with a DFC300FX Digital Color Camera on an inverted Leica DMI4000B scope and processed using the Leica Application Suite (LAS).
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