Fv10 asw 3.0 viewer software

For confocal fluorescent imaging experiments to detect exogenous FA in living HBMECs and HBVPs, cells were treated with 10 μM PFM for 15 min, washed with PBS (pH 7.4), followed without or with the incubation of 200 μM FA for 15 min. For the fluorescence imaging experiments of endogenous FA in living brain cells, HBMECs and HBVPs were incubated without or with 200 μM NaHSO₃ for 30 min, and then washed with PBS (pH 7.4), followed by 10 μM PFM incubation for 15 min. The residual probe was washed three times before imaging. Digital images were captured using the FV10-ASW 3.0 viewer software (Olympus). Cell counts were performed using a 4×, 20×, 40×, or 60× objective in at least five fields of view randomly selected from each coverslip. At least 3 independent experiments were counted. For real-time visualization of endogenous and exogenous FA in HBMECs, cells were incubated with 10 μM PFM after being treated with exogenous FA (500 μM), thapsigargin (5 μM), or NaHSO₃ (200 μM) for 30 min, and fluorescence were obtained with a confocal laser scanning microscope (Olympus, FV1000). For imaging endogenous FA in living brain tissue slides, tissue slides were first incubated without or with 200 μM NaHSO₃ for 30 min, followed by incubation with 20 μM PFM for another 30 min. The fluorescence density was analyzed using Image J software (NIH, Bethesda, MD, USA).