Alexa flour 488 donkey anti rabbit igg

Human aortic valve interstitial cells were seeded into chamber slides (BD Falcon) and Fetuin A starved for 48 h after incubated in serum free DMEM medium. On the day of experimentation, cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and permeated with 0.5% Triton X-100 (Electron Microscopy Sciences). HAVICs were stained with anti-human Fetuin A antibody (Thermo Fisher Scientific, PA5-51594 at 1:500), and Donkey anti-rabbit IgG Alexa Flour 488 (Invitrogen, 710369 at 1:1000). Nucblue reagent and ActinRed 555 reagent (Invitrogen) were used for nuclear and F-actin stain, respectively. Images were obtained using Zeiss LSM 780 confocal microscope.

When cells have reached the desired confluency, remove the media from the dish and add prewarmed (37 °C) staining solution containing MitoTracker^® probe (thermos, Cat# M22425, a working concentration of 200 nM) incubation for 30 min in a CO₂ incubator. Washing the cells. After staining, wash the cells in a fresh, pre-warmed buffer or growth medium. The cells were fixed in 4% paraformaldehyde solution at room temperature for 30 min and were blocked in 3% BSA containing 0.1% Triton X-100 for 1 h at room temperature. Then, the primary antibodie anti-LC3 (CST, Cat# 12741S, 1:200) were used to incubate the samples overnight at 4 °C in a humid chamber, and donkey anti rabbit IgG ALEXA flour 488 (1:1000, #a21206, Invitrogen) secondary antibody were used to incubate the samples for 1 h at room temperature. After three more washes, the stained nuclei were stained with Hoechst 33342 for 20 min. Images were taken using a confocal microscope (Nikon A1RSi, Tokyo, Japan) and analyzed using Software ImageJ (National Institutes of Health).