Immunocytochemistry of Osteoblast Markers

After 14 days of culture, the cells grown on slides were fixed with paraformaldehyde at 4% (PFA 4%) in PBS1x for 30 min. To facilitate the entry of primary antibody into the cell nucleus, the fixed cells were permeabilized with TRITON-X 0.4%. Then, the slides were washed trice with PBS 1X and incubated at room temperature (RT) with PBS-BSA 3% to block aspecific sites. Subsequently, cells were incubated overnight at 4 °C with the diluted (1:200) primary antibody against either RUNX2 (Boster Immunoleader M00442) or COL1A1 (1:50) (PA1-26204, Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, the slides were washed trice with PBS 1X and incubated with secondary antibody diluted 1:1000 (ab150081, abcam and A-21207, Thermo Fisher Scientific) for 30 min at RT. Then, the nuclei were counterstained with Hoechst 33,258 at RT for 5 min [36 (link)]. Finally, the slides were mounted with an anti-fading mounting solution and kept at 4 °C until their visualization with the fluorescence microscope Nikon Eclipse 80i (Nikon, Tokyo, Japan).