Vector s 1000

To localize, within the osteotomies, cells that had initiated differentiation down an osteogenic lineage, immunostaining was performed using standard procedures [18 (link)]. In brief, following deparaffinization, endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 5 min, and then washed in PBS. Slides were blocked with 5% goat serum (Vector S-1000) for 1 h at room temperature. The appropriate primary antibody was added and incubated overnight at 4 °C, then washed in PBS. The primary antibodies used in this study were Osterix (1:1200; ab22552, Abcam, Cambridge, MA, USA) and Cathepsin K (1:200; ab19027, Abcam, Cambridge, MA, USA). Samples were incubated with appropriate biotinylated secondary antibodies (Vector BA-x) for 30 min, then washed in PBS. An avidin/biotinylated enzyme complex (Kit ABC Peroxidase Standard Vectastain PK-4000, Vectorlabs, Burlingame, CA, USA) was added and incubated for 30 min, and a 3,3′-diaminobenzidine (DAB) substrate kit (Kit Vector Peroxidase substrate DAB SK-4100, Vectorlabs, Burlingame, CA, USA) was used to develop the color reaction. Phalloidin immunostaining was performed using Palloidin Control, DyLight 488 conjugate (1:300; PI21833, Invitrogen, Grand Island, NY, USA).

Immunohistochemical studies were performed using cryocut hippocampal sections (coronal, 16 µm) as previously described [28 (link)], with slight changes in blocking. Sections were blocked with either normal chicken serum (Nptx2, Vector S3000, Vector Laboratories, Burlingame, CA, USA) or normal goal serum (Npas4, Vector S1000, Vector Laboratories) for one hour at room temperature. Sections were incubated with different primary antibody concentrations in antibody solution (2.5% BSA in PBS) at 4 °C overnight (Nptx2, 1:100, Abcam, Cambridge, England; Npas4, 1:200, Abcam). Following primary antibody incubation, slides were washed in 1xPBS three times for five minutes each at room temperature. Slides were then incubated with appropriate fluorescent secondary antibody (1:500, Alexa-488 and 594, Invitrogen, Carlsbad, CA, USA) in antibody solution for 1.5 h at room temperature. The slides were then rinsed in 1× PBS three times for five minutes each and cover slipped using VectaMount (Vector Laboratories). Sections were viewed and images were captured using a Nikon Eclipse Ni microscope equipped with DS-Qi1 monochrome, cooled digital camera and NIS-AR 4.20 Elements imaging software. CEPO + IGF-1 and vehicle-treated sections were captured using identical exposure sections. Sections = −3.30 mm from Bregma.

Sections underwent immunostaining procedures to localize cells that had initiated differentiation down an osteogenic lineage within osteotomies [38 (link)]. After deparaffinization, endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 5 min and then washed in PBS. Sections were then blocked with 5% goat serum (Vector S-1000) for 1 h at room temperature, followed by incubation with the primary antibody overnight at 4 °C, and then washed in PBS. The primary antibodies used in this study were Osterix (1:1200; ab22552, Abcam, Cambridge, UK) and Cathepsin K (1:200; ab19027, Abcam). Samples were incubated with corresponding biotinylated secondary antibodies (Vector BA-x) for 30 min; then, they were washed in PBS, and staining was visualized with an avidin/biotinylated enzyme complex (Kit ABC Peroxidase Standard Vectastain PK-4000) incubated for 30 min and a DAB substrate kit (Kit Vector Peroxidase substrate DAB SK-4100).