Mouse anti collagen type 1 antibody

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.

Both the collagen sponge and collagen-PRP sponge were characterized by immunostaining on collagen type I and TGF-β1 according to the published protocol [32 ]. The sponge samples were placed in frozen section medium (Neg 50; Richard-Allan Scientific; Kalamazoo, MI) and solidified completely with liquid nitrogen cold 2-methylbutane. The sponge blocks were cut into 10 μm thick sections and dried overnight at room temperature. The sections were rinsed 3 times with PBS, fixed with 4% paraformaldehyde for 30 min, and further washed with PBS three more times. The sections were coated with 5% goat serum for 30 min at room temperature in a humid chamber. The serum was carefully removed by aspiration, then mouse anti-collagen type I antibody (1:350; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or mouse anti-TGF-β1 antibody (1:350; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were applied, the sections were incubated with the antibodies at room temperature for 2 hrs. The sponge sections were washed three times with PBS, and then reacted with FITC-conjugated goat anti-mouse IgG (1:500; Santa Cruz Biotechnology) for collagen type I and Cy3-conjugated goat anti-mouse IgG for TGF-β1 at room temperature for 1 hr. The sections were washed three times with PBS, and then the positively stained collagen type I (green) and TGF-β1 (red) were checked under a microscope.