Immunohistochemical staining was performed as previously described by Furusho et al. [38 (link)]. After dewaxing and rehydration, the 4.5 μm sections were incubated in 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature (range: 22 ± 5°C) to quench the endogenous peroxidase activity. After incubation with a protein block (DAKO Japan, Tokyo, Japan) for 10 minutes at room temperature, immunolocalization of RANKL, OPG, and cathepsin K was detected using the anti-rat sRANKL rabbit antibody (#ab62516; 1 : 2000 dilution, Abcam, CB, UK) or anti-rat OPG rabbit antibody (#ab73400; 1 : 400 dilution, Abcam, CB, UK) or anti-rat cathepsin K rabbit antibody (#ab19027; 1 : 500 dilution, Abcam, CB, UK). Anti-rabbit IgG antibody (EnVision+ System- HRP Labelled Polymer Anti-Rabbit, Dako Japan) was used as a secondary antibody. After washing the sections twice in phosphate-buffered saline (PBS) for 5 minutes each, staining was visualized using the DAB peroxidase (HRP) substrate kit (Dako Japan) to produce brown reaction products indicative of antigen localization.
Ab62516
Manufactured by Abcam
Sourced in United Kingdom
Ab62516 is a laboratory equipment product. It is a device used for various applications in scientific research and analysis. The core function of this product is to perform a specific task or measurement, but further details on its intended use are not available.
Automatically generated - may contain errors
2 protocols using ab62516
1
Immunohistochemical Analysis of Bone Remodeling Markers
2
Spatial Expression of ICOS, ICOS-L, RANKL
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Immunohistochemistry (IHC) of ICOS, ICOS ligand, and RANKL was performed to visualize the spatial expression of these proteins in paraffin-embedded sections from rat samples.
For IHC, all sections were stained using the R&D HRP-DAB staining kit (CTS017; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The primary antibodies were as follows: ICOS antibody (1:100, ab175401, Abcam), ICOS-ligand antibody (1:100, A7080; ABclonal, Woburn, MA), and RANKL antibody (1:500, ab62516, Abcam). The counterstaining was performed with hematoxylin. The negative controls were treated with phosphate-buffered saline rather than the primary antibodies.
For IHC, all sections were stained using the R&D HRP-DAB staining kit (CTS017; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The primary antibodies were as follows: ICOS antibody (1:100, ab175401, Abcam), ICOS-ligand antibody (1:100, A7080; ABclonal, Woburn, MA), and RANKL antibody (1:500, ab62516, Abcam). The counterstaining was performed with hematoxylin. The negative controls were treated with phosphate-buffered saline rather than the primary antibodies.
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