Dako envision horseradish peroxidase rabbit mouse kit

The tumor cell-specific expression of CYP27A1 was determined by immunohistochemistry following a previously validated protocol (Nelson et al. 2013) . Sections of 3 to 4 µm were cut from whole tissue FFPE blocks, de-paraffinized, treated with antigen retrieval buffer (citrate, pH 6) for 20 min, and then reacted with an anti-CYP27A1 rabbit monoclonal antibody (ab126785, Abcam) at a dilution of 1:500 for 2 h. Staining procedures were performed using the DAKO Envision horseradish peroxidase rabbit/mouse kit (DAKO) and the Dakocytomation Autostainer (DAKO). Cell nuclei were counterstained with hematoxylin. CYP27A1 positivity was detected as a granular cytoplasmic reactivity. Cell type identification and staining intensity was assessed by a board certified pathologist (DG). Only tumor cell-specific CYP27A1 expression was considered for subsequent analyses. Each sample was given a semiquantitative intensity score: 0 (absent), 0.5 (borderline), 1 (weak), 2 (moderate) or 3 (strong). For statistical analysis, the tumors were categorized as negative (0), weak (0.5, 1) and overexpressed (2, 3). Representative cases of CYP27A1 expression in these categories are illustrated in Supplementary Fig. 4.