4 6 diamidino phenyindole dapi

BMSCs were seeded into 24 well plates with slides and incubated at 37 °C with 5% CO₂ and saturated humidity. 48 hours post-transfection, the cells were fixed in ice-cold 4% paraformaldehyde for 30 min. Permeabilization was performed with 0.25% Triton X-100 in PBS for 10 min followed by blocking for 1h with 10% Goat serum. The fixed cells were then incubated with 1:100 dilution of PIWI (Abcam, UK), β-catenin (Santa Cruz Biotechnology, USA), and Wnt2b (Santa Cruz Biotechnology, USA) overnight at 4 °C. After washing three times with PBS, the slides were incubated with fluorescein isothiocyanate (cy3)-conjugated secondary antibody in a 1:200 dilution in PBS for 1 h at room temperature in darkness. The nuclei were subsequently stained with 4,6-diamidino-phenyindole (DAPI) (Abcam, Cambridge, UK) and the slides were then examined using Leica DMi8 microscope (Carl Zeiss MicroImaging GHBH; Jena, Germany). Statistical fluorescence intensity was performed by using ImageJ as the previous study described 36 (link).