Phalloidin Staining and Visualization of Actin

After 14 days of culture on the GelMA-based hydrogels, cells were fixed with 4% paraformaldehyde for 20 min at RT. After rinsing three times with PBS, hydrogels were incubated with Phalloidin-TRITC (2 μg/mL) and Hoechst 33342 (ca. 40 μg/mL) to stain F-actin and cell nuclei, respectively. Hydrogels were incubated with the staining mixture for 60 min at RT while protected from light. Then, they were washed with PBS and placed with the cell layer downward onto a glass coverslip coated with Mowiol. 25× water immersion objective was used for cell imaging.