Hoechst fluorochrome 33342

The stemness of human tendon stem cells (hTSCs) from the patellar tendon used in this study was verified by immunocytochemical analysis of three stem cell markers, including octamer-binding transcription factor 4 (Oct-4), Nanog, and nucleostemin (NS). hTSCs were first seeded into 12-well plates at a density of 20,000 cells/well with 1.5 ml medium and cultured for 3 days. Then, the hTSCs were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature and washed in 0.5% Triton-X-100 in PBS for 15 min. Subsequently, the fixed cells were incubated with either mouse anti-human Oct-4 (1∶500), rabbit anti-human Nanog (1∶500), or goat anti-human nucleostemin (1∶500) overnight at 4°C. After washing three times with PBS, the cells were again incubated for 2 hrs at room temperature with either Cy3-conjugated goat anti-mouse IgG antibodies (1∶1000) for Oct-4, Cy3-conjugated goat anti-rabbit IgG (1∶500) for Nanog, or Cy3-conjugated donkey anti-goat IgG antibodies (1∶500) for Nucleostemin. Nuclei were stained with Hoechst fluorochrome 33342 (1 µg/ml; Sigma, St. Louis, MO). Stained cells were then examined using fluorescence microscopy. All antibodies were obtained from Chemicon International (Temecula, CA), BD Biosciences (Franklin Lakes, NJ), Neuromics (Edina, MN), or Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

The tissue block was cut into 10 μm thick sections, and stained with Alizarin Red S at room temperature for 40 minutes to test bone-like tissue formation. In addition, immunostaining for collagen type I and osteocalcin expression were also performed to verify osteogenesis of BMSCs. Osteocalcin is a noncollagenous protein produced solely by osteoblasts [33 (link)]; hence, it was used as a marker of osteogenesis of BMSCs in this study. The tissue sections were reacted either with mouse anti-collagen type I antibody (1:350; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at room temperature for 3 hours, or mouse anti-osteocalcin antibody (1:350; Abcam, Cambridge, MA) at room temperature for 5 hours. Then the tissue sections were washed 3 times with PBS, reacted with Cy3-conjugated goat anti-mouse IgG (1:500; Santa Cruz Biotechnology, Inc. Santa Cruz, CA) at room temperature for 2 hours, again washed 3 times with PBS, and then reacted with Hoechst fluorochrome 33342 (1 μg/ml; Sigma, St. Louis, MO) at room temperature for 5 minutes. Finally, the sections were washed with PBS twice. Negative control was created by omitting primary antibodies during the immunostaining. The positive stained cells were examined with a fluorescence microscope.

Each frozen tissue block was cut into 10 µm thick sections, placed on glass slides, and then allowed to dry overnight at room temperature. The tissue sections were fixed in 4% paraformaldehyde for 30 min and further washed three times with PBS. They were then incubated at room temperature with mouse anti-human PPARγ antibody (Santa Cruz Biotechnology, Inc., Cat. #271392, Santa Cruz, CA) diluted to 1∶350 for 2 hrs, mouse anti-collagen type II antibody (1∶300; Millipore, Cat. #MAB8887, Temecula, CA) for 2 hrs, or mouse anti-human osteocalcin antibody (1∶300; Santa Cruz Biotechnology, Inc., Cat. #74495, Santa Cruz, CA) for 3 hrs. After washing with PBS, Cy3-conjugated goat anti-mouse IgG (1∶500; Santa Cruz Biotechnology) was added as secondary antibody and incubated at room temperature for 1 hr, followed by staining the nuclei with Hoechst fluorochrome 33342 (1 µg/ml; Sigma, St. Louis, MO) at room temperature for 5 min. Additionally, cell morphology and distribution in those tissues that received hTSCs, which had been treated with various concentrations of PGE₂ in culture, were assessed by staining with hematoxylin and eosin (H&E). Finally, all tissue sections were examined under a fluorescence microscope.