Fitc conjugated goat anti mouse secondary antibody

The phosphorylation of histone H2AX (a marker of DNA double-strand breaks) was analyzed as previously described (15 (link)), with slight modifications. Briefly, 1×10⁵ cells were seeded into 6-well culture plates containing a glass cover slip in each well. Following treatment, the cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS and permeabilized in 0.2% Triton X-100. Following inhibition with blocking serum for 1.5 h, the samples were incubated with a mouse monoclonal anti-H2AX antibody (1:1,000; Cell Signaling Technology, Inc., Boston, MA, USA) for 2 h, followed by incubation with FITC-conjugated goat anti-mouse secondary antibody (1:500; Cell Signaling Technology, Inc.) for 1 h. For staining the nuclei, DAPI was added to the cells and incubated for another 15 min. The cover slip was then removed from the plate, mounted on a glass slide and observed using an Olympus BX53 fluorescent microscope (Olympus, Tokyo, Japan).

HUCs were seeded onto scaffolds and cultured for 7 days. Then, the cells were fixed in 4% paraformaldehyde for 15 min and rinsed three times with PBS for 5 min each. To decrease non-specific background staining, the cells were incubated in 1% BSA for 20 min. The samples were then incubated overnight at 4 °C in mouse pan-cytokeratin antibodies AE1/AE3 (dilution 1:100; Santa Cruz). Subsequently, the samples were rinsed in PBS and incubated in FITC-conjugated goat anti-mouse secondary antibody (dilution 1:100; Cell Signaling Technology (CST)) for 1 h at room temperature. Finally, DAPI was used to label the cell nuclei. The samples were observed under a confocal laser scanning microscope (CLSM) to capture the visual results.