Mouse anti osteocalcin

Immunofluorescence was mainly performed as described^{56 (link)}. Briefly, freshly dissected bones were fixed in 4% paraformaldehyde for 48 h and incubated in 15% DEPC-EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin or OCT and sectioned at 8 μm. Sections were blocked in PBS with 10% horse serum for 1 h and then stained overnight with mouse-anti-Osteocalcin (Santa Cruz, 1:100, sc-376726), rabbit-anti-Ddah1 (SAB, 1:200, #37368), mouse-anti-Ddah1 (Santa Cruz, 1:100, sc-271337), rabbit-anti-Ddah2 (SAB, 1:200, #38934), mouse-anti-TAZ (Abcam, 1:200, ab242313), rabbit-anti-YAP (Abcam, 1:200, ab52771), and eNOS (Santa Curz, 1:200, sc-376751). Goat-anti-mouse FITC (1:1000; Jackson ImmunoResearch, 705-165-147) and donkey-anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) were used as secondary antibodies. DAPI (Cell Signaling Technology, #4083) and DyLight™ 594 Phalloidin (Cell Signaling Technology, #12877) were used for counterstaining. All immunofluorescence experiments were confirmed by at least one independent repeat. An Olympus IX81 confocal microscope or Zeiss LSM-880 confocal microscope was used to image samples.

Fixed and cryosectioned samples were blocked with 3% (w/v) BSA in PBS and permeabilized with 0.3% v/v Triton X-100 for 1 h at room temperature. Samples were incubated with the following primary antibodies in 1% w/v BSA for 1 h at room temperature: mouse anti-osteocalcin (1:200; cat#, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-collagen type II (1:200; II-II6B3-C, DSHB), goat anti-PDX-1 (1:200; 47,383, Abcam, Cambridge, UK), guinea pig anti-insulin (1:200; ab7842, Abcam, Cambridge, UK), and rabbit anti-connexin 36 (1:200; 364,600, Invitrogen, Carlsbad, CA, USA). Samples were washed with PBS and incubated with the following secondary antibodies: anti-rabbit Alexa 555 (1:200; A21428, Thermo Fisher Scientific, Waltham, MA, USA), anti-rabbit Alexa 647 (1:200; cat#, Thermo Fisher Scientific, Waltham, MA, USA), anti-mouse Alexa 488 (1:200; A11001, Thermo Fisher Scientific, Waltham, MA, USA), anti-guinea pig Alexa 488 (1:200; A11073, Thermo Fisher Scientific, Waltham, MA, USA), and anti-goat Alexa 555 (1:200; A31573, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with 2 mg/mL Hoechst 33,342 (1:1000; H21492; Molecular Probes, Eugene, CA, USA) for 10 min at room temperature. Fluorescence images were acquired using a confocal microscope (LSM 710; Carl Zeiss, Oberkochen, Germany) at the Soonchunhyang Biomedical Research Core Facility of the KBSI.

The bone samples (two defects remained in one piece of cranial bone, 15 mm×10 mm approximately) were fixed in 10% formalin-buffered solution at room temperature, then decalcified in 9% formic acid for 3 weeks at room temperature on a rotating rocker. After gradient dehydration in ethanol, the samples were embedded in paraffin and sectioned (thickness = 5 μm) in the transverse plane. Sections were subjected to hematoxylin and eosin (H&E) staining or Safranin O staining.
For immunochemistry staining, the sections were immersed in 10 mM citrate buffer at 60°C for 20 min for antigen retrieval. Then sections were blocked in 5% goat serum and 1% BSA for 30 min, then incubated with the mouse anti-osteocalcin (1:1000, Santa Cruz, USA) or rabbit anti-GFP (1:1000, Santa Cruz, USA) overnight at 4°C. After being washed with PBS for three times, sections were incubated with the goat anti-mouse antibody (1:1000, Santa Cruz, USA) or goat anti-rabbit antibody (1:1000, Santa Cruz, USA) at room temperature for 1 hour. Then, the signal was developed by incubating using the DAB detection system (Dako, USA) for 1 min and observed under a microscope. The osteoid matrix areas were measured using Image J software. Five microscopic fields were chosen randomly from each sample and measured.