Periostin

Following in vitro culture or in vivo implantations, samples were fixed in a solution of 4% paraformaldehyde overnight. Subsequent to fixation, some samples were decalcified in a solution of 0.4 M ethylenediaminetetraacetic acid (EDTA) for 1–2 h. Samples were embedded in paraffin, and 5 μm longitudinal sections were prepared. Sections were stained for hematoxylin and eosin (H&E) to characterize cell and tissue morphology and structure, and un-decalcified sections were also stained for Alizarin Red to detect mineralization. Immunostaining was performed to detect bone sialoprotein (BSP; Millipore); periostin (Novus); periodontal ligament-associated protein 1 (PLAP-1), also known as asporin (Invitrogen); or alkaline phosphatase (ALP; Abcam). Fluorescently tagged secondary antibodies, Alexa Fluor 546 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit (Invitrogen), were used to detect the signal. Negative controls to our immunostaining were performed in parallel by omitting the primary antibody. DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) staining was performed to detect nuclei, and slides were mounted with Immu-mount aqueous mounting medium (Thermo Scientific). Brightfield images were acquired using a Nikon Eclipse TE200-E inverted microscope, and fluorescent images were taken using a Nikon Eclipse Ti inverted microscope.

Antibodies against the following proteins were used: periostin (Novus Biologicals, Centennial, CO, USA; NBP1-30042; 1:300 dilution for IF, 1:1000 for Western blot); collagen I (Abcam, Boston, MA, USA; ab21286; 1:100 for IF); PDGFRα (R&D Systems, Minneapolis, MN, USA; AF1062; 1:100 for IF); collagen 1a2 (Santa Cruz, Dallas, TX, USA; sc-393573; 1:500 for Western blot); TGFb1 (Abcam 215715; 1:1000 for Western blot); TGFb2 (Abcam 205150; 1:1000 for Western blot); TGFb3 (Abcam 15537; 1:1000 for Western blot); pSmad2/3 (MaineHealth, Scarborough, ME, USA; D8591; 1:50 for IF); CD31 (BioLegend, San Diego, CA, USA; 102423; 1:100 for flow cytometry); CD45 (BD Biosciences, Franklin Lakes, NJ, USA; 563890; 1:100 for flow cytometry); MEFSK4 (Miltenyi Biotec, Waltham, MA, USA; 130-120-802; used 1:30 for flow cytometry). Wheat germ agglutinin (488) was from ThermoFisher, Waltham, MA, USA (W11261; 5 µg/mL for immunofluorescence).