Mouse anti alkaline phosphatase alp

To immunostain markers of interest in human BM-MSCs, they were osteo-inducted at the density of 1 × 10⁵ cells/well in 48-well plates (BD Biosciences, San Jose, CA). Fourteen days after osteogenic differentiation, induced cells were washed twice with PBS and fixed with PBS (1×) containing 4% paraformaldehyde at RT for 30 min. Cells were permeabilized with 0.1% Triton X-100 in PBS and then incubated overnight at 4 °C in the presence of mouse anti-alkaline phosphatase (ALP; BD Biosciences, San Diego, US) and collagen type I (ColI; Sigma-Aldrich Chemie, Taufkirchen, Germany) primary antibodies diluted 1:200 and 1:1000 in PBS + 0.1% donkey serum (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. The next day, cells were incubated with Alexa Fluor 568-conjugated donkey anti-mouse IgG (H + L) secondary antibody (Life Technologies Europe, Bleiswijk, the Netherlands) diluted 1:400 in PBS + 0.1% donkey serum for 4 h at RT. Finally, the nuclei were stained with Hoechst 33258 staining (Sigma-Aldrich Chemie, Taufkirchen, Germany) for 10 min. Cells were completely washed with PBS after each step. Images were taken with a fluorescence microscope attached to digital color camera. The Mean fluorescence signal intensity was determined using ImageJ (version 4.1; National Institutes of Health, Bethesda, MA).

Two weeks after osteogenic induction, cells were fixed with 4% paraformaldehyde (in 1× PBS) at 4 °C for 30 min, permeabilized by incubation with 0.1% Triton X-100 in PBS and incubated overnight at 4 °C with mouse anti-alkaline phosphatase (ALP; BD Biosciences, San Diego, CA) and anti-collagen type I (ColI; Sigma-Aldrich Chemie, Taufkirchen, Germany), diluted as 1:200 and 1:1000 in PBS and 0.1% donkey serum, respectively (Santa Cruz Biotechnology, Santa Cruz, CA). The next day, cells were incubated at RT for 4 h with Alexa Fluor 568-conjugated donkey anti-mouse IgG (H + L) secondary antibody (Life Technologies Europe, Bleiswijk, The Netherlands) diluted as 1:400 in PBS and 0.1% donkey serum. Then, nuclei were incubated with Hoechst 33258 (Sigma-Aldrich Chemie, Taufkirchen, Germany) for 10 min at RT. After each incubation step, cells were thoroughly rinsed with PBS. Pictures were taken with an inverse fluorescence microscope (Nikon eclipse TE2000-U) attached to digital color camera. ImageJ, version 4.1 (National Institutes of Health, Bethesda, MA) was used for mean fluorescence signal intensity.