Ab197375

Immunohistochemistry (IHC) analyses were performed as described previously [21 (link)]. Antibodies against AR (ab9474; dilution, 1:200), PDEF (ab197375; dilution, 1:200), MAD1 (ab175245; dilution, 1:200) and MYC (ab32072; dilution, 1:200) were purchased from Abcam. Anti-Ki67 antibody (sc-23,900; dilution, 1:200) was purchased from Santa Cruz Biotechnology. Normal breast tissue sections were processed simultaneously and were used as positive controls for AR and PDEF, and normal goat serum-substituted primary antibodies were used as negative controls. Two senior pathologists independently quantified IHC slides. IHC scores of PDEF were used to the multiplied result of percentage positivity and staining intensity in the stained tissue area, and total scores ranged from 0 to 6. Percentage positivity was scored as 0 (0–25%), 1 (26–50%) and 2 (> 50%), and staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). A total score of ≥0 and ≤ 3 indicated negative PDEF expression, and a total score of ≥4 indicated positive PDEF expression [21 (link)]. AR expression was considered to be positive if nuclear staining was observed in > 10% tumour cells.

The following antibodies were used: Polyclonal rabbit antibody against human myeloperoxidase (MPO) (A0398, Dakocytomation, Glostrup, Denmark); polyclonal rabbit anti-muc2c3 (kindly provided by the University of Gothenburg, Gothenburg, Sweden); polyclonal rabbit anti-SAM-pointed domain containing ETS transcription factor (SPDEF) (ab197375, abcam, Cambridge, United Kingdom); monoclonal rabbit anti-BiP (C50B12, cell signaling technology, Leiden, The Netherlands); monoclonal mouse anti-CHOP (clone 9C8, MA1-250, Thermo Fisher Scientific, Waltham, MA, USA), and polyclonal rabbit anti-cleaved caspase-3 (CC3) (asp175) (9661, cell signaling technology, Leiden, The Netherlands).
Secondary antibodies used were: Peroxidase-conjugated goat anti-rabbit (111-035-045, Jackson, West Grove, PA, USA), biotin-conjugated rabbit anti-mouse (E0413, DakoCytomation, Glostrup, Denmark), and biotin-conjugated swine anti-rabbit (E0353, DakoCytomation, Glostrup, Denmark).