Goat anti rabbit tritc

Osteocytic populations in isolated and cultured cells were characterized for sclerostin production. Cells were fixed as described earlier and permeabilized using 0.1% (v/v) triton x-100 for 10 min. To block unspecific antibody binding, cells were incubated in PBS with 3% (w/v) BSA for 1 h at room temperature. The cells were then further incubated overnight at 4°C with a rabbit anti-human sclerostin antibody (Abcam) followed by secondary staining with a fluorescence-conjugated antibody (goat anti-rabbit TRITC, Abcam). Cells were also stained with DAPI. Early osteocytic cell line (MLO-A5) used as ALP-positive and sclerostin-negative control. Sclerostin-positive cells were counted in 10 different randomly selected areas containing between 100–500 cells per field.

The 3D culture samples were removed from the culture chambers and fixed with 4% PFA. The fixed samples were paraffin-embedded, cut into 10-μm thick histological sections, and stained with hematoxylin and eosin (H&E, Sigma-Aldrich). 20 cells from 3 separate histology images were randomly selected at the center of 3D tissue. The nucleus-to-nucleus distance was measured from the selected cells with the nearest interstitial space between beads. Slides were deparaffinized and permeabilized using 0.1% (v/v) triton x-100 for 10 min. To block unspecific antibody binding, cells were incubated in 3% (w/v) BSA made in PBS for 1 h at room temperature. The cells were then further incubated overnight at 4 C with a rabbit anti-human sclerostin antibody (Abcam) followed by secondary staining with a TRITC-conjugated antibody (goat anti-rabbit TRITC, Abcam). Cells were counterstained with DAPI (Sigma-Aldrich) and mounted. Slides stained with secondary antibody only were used as negative control. The stained slides were observed under a fluorescence microscope (Eclipse Ti-E, Nikon).
To determine the numbers of sclerostin positive cells upon PTH treatment and mechanical loading compared to control culture, 3 separate images per condition were selected and sclerostin expression was quantified in 30 to 50 cells in the center of 3D tissue.