Goat anti sclerostin antibody

For sclerostin staining, sections of alveolar bone were deparaffinized and rehydrated through a series of graded ethanol solutions. To inhibit endogenous peroxidase activity, sections were quenched in 3% H₂O₂ for 20 min and then treated with trypsin (Thermo Fisher Scientific, Waltham, MA, USA) for antigen retrieval. Immunohistochemistry was carried out using universal kits (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Sections were preincubated with normal horse blocking solution for 20 min and then incubated overnight at 4 °C with goat anti-sclerostin antibody (1:300 dilution, R&D Systems, Minneapolis, MN, USA). Sections were developed using the 3,3′-diaminobenzidine chromogen and counterstained with methyl green. Slides were scanned using a Aperio AT2 Digital Whole Slide scanner (Leica Microsystems Inc., Buffalo Grove, IL, USA) and analyzed using Aperio ImageScope software (version12.3.2.2013, Aperio Technologies Inc., Vista, CA, USA). Numbers of sclerostin-positive osteocytes were counted in the ROI, which extended 0.5 mm from the ABC in the furcations of the first molars, and are presented as the percentage of sclerostin-positive osteocytes per number of total osteocytes in the ROI.

To assess sclerostin expression in tibiae, tibiae sections were deparaffinized in xylene and rehydrated through ethanol series. Sections were incubated with Trypsin Enzymatic Antigen Retrieval Solution (Abcam, Cambridge, UK) for 15 min at 37 °C followed by immersion in 3% H₂O₂ in methanol for 20 min to block endogenous peroxidase. After blocking in normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 20 min, the sections were incubated overnight at 4 °C with goat anti-sclerostin antibody (1:50 dilution, R&D Systems, Minneapolis, MN, USA). After incubation with anti-goat secondary antibody (Vector Laboratories) for 20 min, sections were developed with 3,3-diaminobenzidine tetrahydrochloride substrate chromogen system (DAKO, Botany, Australia) and counterstained with methyl green. Slides were scanned as described above. The number of sclerostin-positive osteocytes in the ROI was divided by the number of total osteocytes in the ROI. The ROI was the same as the ROI for osteoclast counting.

Whole bones (tibia) from control and diabetic rats were fixed in 10% formalin for 24 hours, then decalcified for 2–3 weeks in 14% EDTA. Bone sections were prepared as described above. These were dewaxed and rehydrated prior to immunostaining. Sections were then incubated in blocking solution, followed by goat anti-sclerostin antibody (1:250, R&D Systems Inc., Minneapolis, MN) overnight at 4 °C. Cy3-donkey anti-goat antibody (Jackson ImmunoResearch, Burlingame, CA) was then added after rinsing slides and incubated for 1 hour at room temperature. Slides were incubated with DAPI (Sigma) for 30 minutes at room temperature, rinsed and cover-slipped and imaged with a laser confocal microscope (Carl Zeiss Microscopy GmbH, Munich, Germany). Cell counting was performed as an average of 9 images of diaphyseal bone obtained from standardized regions (proximal, middle and distal regions from each cortex) under 25x oil magnification in blinded fashion. Percent positive cells were expressed as number of positively stained cells over the total number of DAPI-positive cells times 100. A minimum of two sections per bone were used for each specimen.