Zeis lsm900 confocal laser scanning microscope

Scaffolds were fixed in 10% formalin, paraffin-embedded, and sectioned at 4 µm. Following deparaffinization, the sections were treated with 0.5% Triton X-100 (MP Biomedicals, LLC, Santa Ana, CA), blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) for 1 hour, and subjected to heat-induced antigen retrieval using sodium citrate buffer(10 × 10⁻³m, pH 6, Fisher Scientific International, Inc., Pittsburgh, MA) at >80 °C, for 20 min. Sections were then separately incubated with anti-p-Smad1/5 (1:500, Cell Signaling Technologies), anti-Yap (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-non-p-β-catenin (1:1000 Cell Signaling Technologies, Beverly, MA) overnight in 4 °C. After washing, sections were incubated in anti-rabbit or anti-mouse IgG Alexa Fluor Plus 594 (Thermo Fisher, Eugene, OR). Coverslips were mounted with Prolong Gold Antifade Reagent with Dapi (Cell Signaling Technologies, Danvers, MA). Images were captured with the Zeiss Axio Observer 3 inverted microscope with the ZEN 2.3 Pro software (Zeiss, Oberkochen, Germany) and the Zeis LSM900 confocal laser scanning microscope with Zen 3.1 Blue software (Zeiss, Oberkochen, Germany).

Scaffolds were fixed in 10% formalin, paraffin-embedded, and sectioned at 4 μm. Following deparaffinization, the sections were treated with 0.5% Triton X-100 (MP Biomedicals, LLC, Santa Ana, CA), blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) for 1 hour, and subjected to heat-induced antigen retrieval using sodium citrate buffer(10 mM, pH 6, ThermoFisher, Waltham, MA) at >80°C, for 20 minutes. Sections were then separately incubated with anti-Yap (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) anti-non-p-β-catenin (1:1000 Cell Signaling Technologies, Beverly, MA) overnight at 4 °C. After washing, sections were incubated in anti-rabbit IgG Alex Fluor Plus 488 or anti-mouse IgG Alexa Fluor Plus 594 (ThermoFisher, Waltham, MA). Coverslips were mounted with Prolong Gold Antifade Reagent with Dapi (Cell Signaling Technologies, Danvers, MA). Images were captured with the Zeis LSM900 confocal laser scanning microscope with Zen 3.1 Blue software (Zeiss, Oberkochen, Germany).

For in vitro osteoclast cultures, 8 mm scaffolds were co-cultured with primary human osteoclasts on chamber slides (Lab-Tek, ThermoFisher, Waltham, MA) for 14 days as described above. Scaffolds were then removed from the chamber slides and slides were fixed with 4% paraformaldehyde. Slides were then blocked and permeabilized with PBS-Triton 0.02% plus 10% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour and subjected to heat-induced antigen retrieval using sodium citrate buffer (10 mM, pH 6) at >80°C, for 20 minutes. Slides were then incubated with anti-TRAP (AbCam, Waltham, MA; 1:100) overnight at room temperature. After washing, slides were incubated in anti-mouse IgG Alexa Fluor Plus 594 (Cell Signaling Technology, Danvers, MA; 1:1000) for 4 hours. After washing again, slides incubated with Alexa Fluor ® 488 phalloidin (Cell Signaling Technology, Danvers, MA, 1:50) to label actin for 45 minutes. Slides were then washed and incubated with Dapi (Cell Signaling Technology, Danvers, MA; 1:1000) for 10 min. Coverslips were mounted with Prolong Gold Antifade Reagent (Cell Signaling Technologies, Danvers, MA). Images were captured with the Zeiss Axio Observer 3 inverted microscope with the ZEN 2.3 Pro software (Zeiss, Oberkochen, Germany) and the Zeis LSM900 confocal laser scanning microscope with Zen 3.1 Blue software (Zeiss, Oberkochen, Germany).