Cool snap pro color digital camera

For measurement of antioxidant activities in the zebrafish, the embryos (3 dpf) were transferred to a 12-well plate. The embryos were treated with DCFH-DA (20 μg/mL), and incubated in the dark room for 1 h at room temperature for ROS measurement. The embryos were washed using fresh medium and anesthetized using Ethyl 3-aminobenzoate methanesulfonate (Sigma-Aldrich, St. Luis, MO, USA) before observation. We placed each anesthetized larva on the slide glass, and fluorescence values were measured using a fluorescence microscope. The differences in the fluorescence intensities of the groups were noted by keeping the value of the control group fixed as the index for the comparisons. Lipid peroxidation was measured by treatment with diphenyl-1-pyrenylphosphine (DPPP, Thermo Scientific, USA). Similarly, embryos in the well plates were treated with DPPP (25 μg/mL) and incubated. Washing and anesthetizing was carried out, as mentioned above. Cell death was measured by treatment with acridine orange (Sigma-Aldrich, St. Louis, MO, USA), (7 μg/mL). The observation of zebrafish was conducted using Cool SNAP-Pro color digital camera (Olympus, Japan) according to Kim et al [13 (link)]. The fluorescence intensities of the zebrafish larvae were quantified using Image J (Version 1.51) program.

The ROS production of the zebrafish was analyzed using an oxidation-sensitive fluorescent probe dye, 2,7-dichloro-dihydro fluorescein diacetate (DCFH-DA). The DCFH-DA was deacetylated intracellularly by nonspecific esterase, which was further oxidized to the highly fluorescent compound dichlorofluorescein (DCF) in the presence of cellular peroxides [55 (link)], At 4 dpf, the zebrafish larvae were transferred to one well of a 24-well plate, treated with DCFH-DA solution (20 µg mL⁻¹), and incubated for 1 h in the dark at 28.5 ± 1 °C. After incubation, the zebrafish larvae were rinsed with fresh embryo media and anesthetized by 2-phenoxy ethanol (1/500 dilution sigma) before observation and photographed under the microscope CoolSNAP-Pro color digital camera (Olympus, Tokyo, Japan). The fluorescence intensity of individual zebrafish larvae was quantified using the Image J application.

In the zebrafish larvae that were exposed to PS14, ROS generation was evaluated using the 2,7-dichloro dihydro fluorescein diacetate (DCFH-DA) assay [38 (link)]. From the in vivo toxicity experimental group, three larvae (96 hpf) were randomly taken from each group. The larvae were euthanized, fixed in 4% paraformaldehyde and stained with 20 µg/mL DCFDA for 20 min in the dark. Stained embryos were washed with PBS and observed under a fluorescence microscope equipped with a Cool SNAP-Pro color digital camera (Olympus, Tokyo, Japan). Results were recorded as photomicrographs, and the fluorescence intensity was quantified using ImageJ software (V.1.49, NIH, Bethesda, MD, USA).

Apoptotic cells were identified by TdT-FragEL DNA Fragmentation Detection Kit (Cat #QIA33, Calbiochem Biosciences, La Jolla, CA, USA). CA1 and CA3 hippocampus histological sections were processed as follows: The sections were rinsed with Tris-buffered saline 0.02 M pH 7.6 (TBS) and incubated with proteinase K (20 μg/mL) for 10 min and endogenous peroxidase was blocked. Afterwards, they were incubated with terminal deoxynucleotidyl transferase enzyme (TdT), which catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides. Biotinylated nucleotides were detected using a streptavidin-peroxidase conjugate and visualized with DAB-urea solution with metal enhancer. Sections were counterstained with 0.1% methyl green and mounted with synthetic resin. The slides were examined with an Olympus IX-71 Inverted Microscope and photographed with a CoolSnap-Pro Color Digital Camera. The percentage of apoptotic cells was obtained by counting the number of positive cells from a total of one hundred cells in three different fields (200×). The CA1 and CA3 regions of the hippocampus were evaluated.

The protective effect of IOC against H₂O₂ was investigated using approximately 7–9 h post-fertilization (hpf) embryos by treating IOC (50, 100, and 200 μg·mL⁻¹); controls were untreated. Following treatment for 1 h, the embryos were stimulated with H₂O₂ (5 mM), and the plate was incubated for 3 dpf. The survival rate and heart beating rate were counted according to the previously optimized method [30 (link)]. Acridine orange and DCFH-DA were utilized to evaluate the cell death and intracellular ROS generation, respectively [31 (link)]. Zebrafish larvae were photographed using a fluorescence microscope (CoolSNAP-Pro Color Digital Camera; Olympus, Japan) and the fluorescence intensity was quantified using ImageJ software (version 1.50i, NIH, USA).

Based on a previous study, cell death, NO production, and intracellular ROS levels were measured using acridine orange (AO), DAF-FM-DA, and DCFH-DA staining, respectively. Following staining, larvae from the zebrafish were rinsed twice using embryo culture media to remove excess stain and anesthetized with 2-phenoxyethanol. Observations were carried out with a microscope equipped with the Cool SNAP-Pro Color Digital Camera (Olympus, Tokyo, Japan). The ImageJ program was used to perform the quantitative measurements of zebrafish larvae [34 (link)].

As in the procedure described by Naito et al., the apoptosis body formation was assessed by nuclear staining [46 ]. After being seeded in 24-well plates for 24 h, cells were treated with LJSF4 and incubated for another 2 h. Then, cells were exposed to 30 mJ/cm² of UVB and incubated with serum-free DMEM solution for 6 h. After incubation, cells were treated with Hoechst 33342 (stock, 10 mg/mL) for 10 min. At last, the stained cells were photographed by a fluorescence microscope equipped with a Cool SNAP-Procolor digital camera (Olympus, Tokyo, Japan). Apoptosis levels were measured using Image J software automatically.