Bz x800 fluorescence microscope

HUVECs (C-12200, PromoCell) were incubated with low serum growth supplement (LSGS) supplemented media for 5 days. Next, cells were harvested and resuspended in non-supplemented media in the presence of ATG (0.05 mg/mL and 0.1 mg/mL), CTLA4Ig (0.5 mg/mL and 1 mg/mL), or a combination of CTLA4Ig 1 mg/mL and ATG (0.05 mg/mL or 0.1 mg/mL). Supplemented media and Suramin (30 µM, Sigma) were used as positive inducer and inhibitor of tube formation, respectively. Non-supplemented media was used as control (Veh). Matrigel was distributed in a 24-well plate and incubated for 30 min at 37 °C to permit gelling. HUVECs were prepared at a density of 4.1 × 10⁵ cells/mL and 300 µL of cell suspension was added into each well. After overnight incubation at 37 °C, cells were stained with Calcein AM (Invitrogen) and tube formation assessed using a Keyence BZ-X800 Fluorescence Microscope (Keyence). Four pictures per well were taken using the 4X objective and GFP filter. Images were analyzed using the Angiogenesis Analyzer plugin in ImageJ software.

The phenotypic features of cell death were evaluated by use of Hoechst33342/PI double staining assay. After treatment for 48 h with different concentrations (3, 10, 30 and 100 nM) of Helle, MCF-7 cells were washed twice with cold PBS, followed by incubation with 100 μl of staining solution (3 μl/ml of PI and 0.05% Hoechst in PBS) for 15 min at room temperature. The staining images were captured using a BZ-X800 Keyence fluorescence microscope (Keyence, Osaka, Japan) and Leica X software at 100×magnification (Leica, Tokyo, Japan).

HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) and HER2-negative breast cancer cells (MCF7) were seeded on a glass-bottom 96-well plate. To test the specificity of the conjugate binding, the HER2 Affibody-IR700Dye conjugate (1 µM) and the trastuzumab-Alexa488 (1 µM) conjugate were added to the media and the cells were incubated for 30 min at 37 °C. After washing the cells with PBS, the cells were examined using a BZ-X800 fluorescence microscope (Keyence, Osaka, Japan). To overlay images, ImageJ was used [38 (link)].

In Experiment 3, brain samples were harvested from rats survived for 72 h. Neuronal degeneration was evaluated using Fluoro-Jade B (FJB) staining, as described previously [25 (link),26 (link)]. Briefly, following the 72-h survival in rats subjected to 6–12 min CA, animals were anesthetized with isoflurane, perfused with saline via the left ventricle, and then decapitated. The brains were fixed with 4% paraformaldehyde at 4 °C. Tissues were embedded in OCT Compound (Tissue-Tec, Sakura Finetek USA, Inc., Torrance, CA, USA), frozen in liquid nitrogen, cut serially with 10 µm thickness in a cryostat, and collected on glass slides. Three microscopic fields (at 40× magnification) were selected from the cerebral cortex, hippocampus, and caudoputamen by an investigator who was blinded to the experimental groups. The investigator selected arbitrary points that were considered to show the representative images of the entire area of each brain region. The number of FJB-positive cells was counted by an investigator who was blinded to the experimental groups (n = 6, 4, and 5 for 6, 9, and 12 min CA, respectively). Images were captured using a BZ-X800 fluorescence microscope (Keyence Corporation of America, Elmwood Park, NJ, USA).

The tumor‐targeting effect of PAZA was evaluated in both WT/ITGA5 KO LLC model (s.c). and the orthotopic model using a larger PVD carrier as a control. Hydrophobic fluorescence dye DiR was loaded into the PAZA carrier or PVD control polymer at a wt/wt ratio of 40:1 and intravenously injected into the mice for real‐time imaging at the DiR dosage of 1 mg k⁻¹g. After 24 h, the mice were imaged by an IVIS 200 system with excitation at 730 nm and emission at 835 nm. The tumor and various organs were then excised for ex vivo imaging following our previous protocol.^[31 (link)
^] The tumor was then frozen sectioned, and stained with DAPI to label the cell nucleus and the antibody for CD31 to label the vascular endothelial cell. The fluorescence signals in the core of the tumor were examined under Keyence BZ‐X800 fluorescence microscope.

DNA repair capability after various treatments was evaluated by determining the RAD51 foci via immunofluorescence staining. Cells after various treatments were washed, fixed, and permeabilized in 0.25% Triton/PBS. Blocking was performed in 5% BSA/0.1% Triton/PBS, followed by incubation with primary antibodies in dilution buffer (1%BSA/0.1%Triton/PBS) (overnight, 4 °C). Cells were then washed in PBS and incubated with secondary antibodies, followed by washing and mounting on coverslips using ProLong Gold antifade reagent with DAPI. Cells were imaged using Keyence BZ‐X800 fluorescence microscope and analyzed using ImageJ/Fiji software. FindFoci ImageJ plugin was used for RAD51 foci quantification, at least five fields were counted for foci number respectively.^[38 (link)
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