Ab9474

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.

Rabbit polyclonal anti-Ki-67 antibody (RB-1510-P) was from Thermo Scientific. Rabbit polyclonal anti-EGFR antibody (ab2430), rabbit anti-smooth muscle actin alpha polyclonal antibody (ab5694), p63 polyclonal antibody (ab53039), p40 polyclonal antibody (ab167612), AR clonal antibody (ab9474), rabbit monoclonal EGFR antibody (ab52894) and phosphoY1068 EGFR antibody (ab40815) were from Abcam. Rabbit anti-elastase I polyclonal antibody was from Life Span BioSciences, Inc. Biotin rat anti-mouse CD45 (53078), rat anti-mouse Ly-6G and Ly-6C (Gr-1) (550291) were from BD Biosciences and anti-mouse F4/80 (14-4801) was from eBioscience. Antibodies against AKT (9272), phosphor-S473 AKT (9271), and phosphor-ERK1/2 (4370) were from Cell Signaling. Anti-ERK1 antibody (sc-93) was purchased from Santa Cruz and anti-ACTIN antibody (MAB 1501R) from Millipore.

Chromatin immunoprecipitation (ChIP) was carried out as described previously following treatment with cAMP (0.5 mM) for fertile controls (15 (link), 60 (link)). Following treatment, hESC cells were fixed using 1% formaldehyde solution (Sigma^®), quenched with 2.5M Glycine (Sigma^®) and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions. The pellet was then sent to Active Motif for sequencing. An anti-WT1 antibody (ab89901 Abcam, UK) and anti-androgen Receptor antibody (ab9474, Abcam, UK) was used to probe for WT1 and AR-target region enrichment respectively.

IHC for AR (ab9474, Abcam), FGFR1 (9740, CST), P-ERK (4370, CST), SOX2 (3579, CST), CHGA (ab45179, Abcam), SYP (ab52636, Abcam) and ARHGEF2 (ab155785, Abcam) was performed using PV-6000 system (ZSGB-BIO, China). Briefly, the slides were immersed in 1X Tris-EDTA (pH 9.0) buffer (Solarbio, China) and placed in a microwave oven for 10 min on high heat, and then adjusted to medium-low heat for 10 min to restore the antigen. 3% H₂O₂ was added to remove endogenous peroxidase in tissue samples. Cover the tissue on the slide with the primary antibody, place it in a humid box, and incubate overnight at 4 °C. After rewarming at room temperature for 30 min, horseradish peroxidase-linked secondary antibody (ZSGB-BIO, PV-6000, China) was added to the specimen and incubate the slides at room temperature for 30 min. After being stained with the DAB solution, the slides were immediately placed in water to stop dyeing, slides were subsequently counterstained with hematoxylin. The tissue is then dehydrated and preserved with neutral balsam (OriGene, ZLI- 9555, China).

IHC was performed for AR as described (14 (link)). Briefly, cells were seeded on coverslips, cultured, and treated as indicated. Following treatment cells were fixed, permeabilized, and blocked overnight. AR was visualized using primary anti-AR antibody (RRID:AB_307266; #ab9474; Abcam) and CD31 (RRID:AB_726362; #ab28364; Abcam). ProLong Gold Antifade Reagent with DAPI (#P36941, Thermo Fisher Scientific) was used to visualize the nuclei. Cells were viewed under a Nikon Eclipse Ti-S fluorescent microscope and images captured using NIS-Elements software.

ERα, ERβ and AR expression was detected by immunohistochemistry using UltraSensitive™ SP kit (#9710, Maixin, Fuzhou, China) according to the manufacturer's instructions. In brief, the sections were deparaffinized, rehydrated and subjected to antigen retrieval (citrate buffer, pH=6.0). The sections were then incubated overnight at 4°C with the primary mouse monoclonal antibodies to ERα (clone 33, ab2746, Abcam; 1:50), ERβ (clone 14C8, ab288, Abcam; 1:100) and AR (clone AR 441, ab9474, Abcam; 1:200), respectively. The sections were subsequently washed and incubated with a secondary antibody. Reaction products were visualized with 3, 3’diaminobenzidine tetrahydrochloride and counterstained with hematoxylin and eosin.

IHC for AR (ab9474, Abcam), FGFR1 (9740, CST), P-ERK (4370, CST), SOX2 (3579, CST), CHGA (ab45179, Abcam), SYP (ab52636, Abcam) and ARHGEF2 (ab155785, Abcam) was performed using PV-6000 system (ZSGB-BIO, China). Briefly, the slides were immersed in 1X Tris-EDTA (pH 9.0) buffer (Solarbio, China) and placed in a microwave oven for 10 min on high heat, and then adjusted to medium-low heat for 10 min to restore the antigen. 3% H₂O₂ was added to remove endogenous peroxidase in tissue samples. Cover the tissue on the slide with the primary antibody, place it in a humid box, and incubate overnight at 4 °C. After rewarming at room temperature for 30 min, horseradish peroxidase-linked secondary antibody (ZSGB-BIO, PV-6000, China) was added to the specimen and incubate the slides at room temperature for 30 min. After being stained with the DAB solution, the slides were immediately placed in water to stop dyeing, slides were subsequently counterstained with hematoxylin. The tissue is then dehydrated and preserved with neutral balsam (OriGene, ZLI- 9555, China).