Ix70 inverted fluorescence phase contrast microscope

Hoechst 33,258 is a fluorescent dye that binds to the regions of DNA rich in adenine–thymine. It forms with DNA a complex, which is brightly fluorescent, and can be easily detected by fluorescent microscope. HK-2 cells were seeded on glass coverslips in 6-well plates and treated with various concentrations of AA I with or without genipin for 24 h. After treatment, the cell medium was discarded and cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at 37 °C. Then the fixed cells were washed with PBS and stained with Hoechst 33,258. The cells were then incubated in the dark for 5 min at 37 °C, and photographed with a fluorescence microscope (Olympus IX70 Inverted Fluorescence Phase Contrast Microscope, Tokyo, Japan).

Immunofluorescence staining was performed as previously described (Chen et al., 2020 (link)). Briefly, NSCs were fixed with 4% paraformaldehyde and treated with 0.1% Triton X-100 for 30 min. Cells were blocked in 5% normal goat serum for 1 h, and the primary anti-mouse Nestin antibody (ab105398, Abcam) was added into the cells for overnight incubation at 4°. The cells were washed and further incubated with goat anti-rabbit immunoglobulin G (IgG) antibody (ab150077, Abcam) at 37° for 1 h. The cells were sealed using glycerol and were examined using an Olympus IX70 inverted fluorescence phase contrast microscope.