Spot ccd camera

Isolation of rat aortic endothelial cells (ECs) from primary explants was prepared from male Sprague-Dawley rats (4 weeks of age) as previously reported 35 (link). Pure endothelial cells were maintained with 10 % FBS/DMEM at 37°C in an incubator with a humidified atmosphere of 5 % CO₂. The confluent cell at passage numbers 3-6 exhibited a typical “cobblestone” growth pattern 35 (link), which identified with the endothelium-specific antibody, von Willebrand Factor (vWF) 36 (link) were used for the experiments. A density of 4 x10⁵ cells/mL seeded into 10-cm plates were treated with vehicle (normal saline) or t-BHP (50, 100, 250, 400, or 1000 μM) in 2 % FBS/DMEM for 15 min, 30 min, 1 hour, 2 h, or 24 h according to previous studies 10 (link),18 -19 (link),35 (link),37 (link).
Changes of cell morphologies were observed under the CKX41 inverted phase-contrast light microscope (Olympus, Japan) and the digital images were captured by Spot CCD Camera which driven by Advanced Spot RT Software version 3.3. The cell viability in response to t-BHP treatment was assessed by MTT assays as describe by Yeh et al. 38 . The optical density (O.D.) values reflected the MTT reductase activities and hence the amounts of viable cells. Results were expressed with respect the control. At least three experiments were conducted for each treatment, using the mean value for data analyses.

Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) were performed as described by Kato et al. (2004) (link) and Han et al. (2006) (link). The labeled genomic DNA of T. elongatum was used as a probe for GISH analysis. Oligo-pAs1-1, Oligo-pAs1-3, Oligo-pAs1-4, Oligo-pAs1-6, Oligo-AFA-3, Oligo-AFA-4 (red), Oligo-pSc119.2-1, and (GAA)₁₀ (green) probes were used according to Du et al. (2017) (link). Oligonucleotide probes were synthesized by Shanghai Invitrogen Biotechnology Co. Ltd. (Shanghai, China). The slides were mounted in Vectashield antifade solution containing 4′-6-diamino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, United States). A fluorescence microscope (BX60, Olympus Corp., Tokyo, Japan) fitted with a Spot CCD camera was used to capture hybridization signals. The images were compiled with CellSens Vers.1.5 Imaging software (Olympus Corp.).

Undifferentiated neutrospheres were maintained in DMEM/F12 (1:1) serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). For differentiation, neutrospheres were dissociated into single cells and a monolayer culture was established by culturing the single cells on at a density of 2 × 10⁴ cells/cm² onto pre-coated Lab-Tek chamber slides (NalgeNunc, Rochester, NY, USA), previously treated with Matrigel Matrix. The cells were differentiated into neurons, astrocytes and oligodendrocytes by culturing into a differentiation medium in which FGF and EGF were replaced with fetal bovine serum (FBS) at 1%. The cells were fixed in with 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with Tris-HCl (0.1 M) + TritonX100 (0.25%) for 10 min, blocked with 10% normal goat serum and then incubated overnight at 4 °C with the primary antibody (anti-nestin, anti-glial fibrillary acidic protein, and anti-MAP2). Following washing, the cells were incubated with specific secondary antibodies for one hour at room temperature. The nuclei were stained with bisBenzimide H33258 diluted in PBS (0.2 µg/mL; SIGMA, St. Louis, MO, USA). Immunocytochemistry analysis was performed using fluorescent microscopy on an OLYMPUS Bx5 with Spot CCD Camera (Olympus Corporation BX 60 Fluorescence Microscope, Shinjuku, Tokyo, Japan).