Sc 67261

For western blot, frozen tissues were homogenized in T-Per (Thermo Fisher) and quantitated with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher). Sixty micrograms of protein were electrophoresed in an SDS-polyacrylamide gel, blotted and probed with antibodies to the following targets: CYP27B1 (sc-67261, Santa Cruz Biotechnology) and GAPDH (Cell Signaling). Densitometry was done with the VersaDoc Imaging System (Bio-Rad) and quantified using Image Lab (Bio-Rad). Densitometry values for CYP27B1 were normalized to those of the housekeeping control (GAPDH) for each lane. Three normal controls were run on each blot and CYP27B1 values for disease samples were expressed as a fold-difference from the mean value of the normal samples. For immunohistochemistry, 10 micron paraffin sections were used. Deparaffinized sections were immersed in 10 mM citrate buffer (pH 6.0) heated at 100°C for 30 min, and allowed to cool to room temperature. After antigen retrieval, all sections were treated with 3% H2O2 for 10min. After blocking, sections were incubated with CYP27B1 antibodies (ab95047, Abcam) overnight at 4°C. Slides were incubated for an hour at room temperature with OneStep polymer HRP anti-rabbit secondary antibody (GTX83399, Genetex, Irvine). After rinsing with PBS, slides were developed with a DAB peroxidase kit (Vector Laboratories) and counterstained with hematoxylin.