Anti ar antibody

IHC staining was performed on sections of paraffin-embedded tissues. All formalin-fixed prostate tissues were embedded in paraffin, and 5-µm sections were prepared. Sections were processed by heating at 70℃ in an oven for 30 min, dewaxing with xylene and alcohol (2 cycles, 10 min each), deparaffinization, and rehydration. Then endogenous peroxidase was blocked for 20 min in 0.3% hydrogen peroxide in water. The slides were then treated for antigen retrieval in a citrate buffer (pH 6) for 10 min at 95°C (DAKO PT Link, Glostrup, Denmark). The primary antibodies were anti-ESM1 (Abnova, H00011082-M02) and anti-AR antibody (Santa Cruz, sc-815). Samples were incubated with DAKO envision that contained horseradish peroxidase conjugated goat anti-rabbit, goat anti-mouse, or rabbit anti-goat antibodies (DAKO Cytomation, Glostrup, Denmark) for 30 min. The slides were visualized using a diaminobenzidine solution (DAB+; DAKO kit).

VCaP or VCS2 cell were treated with 10 nM of DHT for 4hr, followed by ChIP-Seq analyses as previously described.^{47 (link)} Briefly, ChIP was carried out using anti-AR antibody (Santa Cruz, Dallas, TX, USA, Sc-816 ×), followed by libraries construction and sequencing using the Illumina Genome Analyzer (San Diego, CA, USA). Significantly enriched regions (P<10⁻¹⁵ or <10⁻⁵) were detected using the MACS software (2.0) with default parameters. The Gene Expression Omnibus (GEO) accession number for the ChIP-Seq analyses is GSE32345.
For preparation of ChIP-qPCR, dispensed cells were formalin fixed, lysed, and sonicated to break the chromatin into 500–800 bp fragments, followed by immunoprecipitation. The qPCR was carried out using SYBR Green (Thermo Fisher Scientific, Waltham, MA, USA). The primers are listed as following: ACSL3-ARE: forward, 5′-GAGTTGTCATCCTGGGCACT-3′, reverse, 5′-GGGGCCTGATTATTGGGTAT-3′ MBOAT2-ARE: forward, 5′-GTAGGTTTGGACTGGCAGCA-3′, reverse, 5′-CGTAGCACCACGCATTACTC-3′ ELOVL7-ARE: forward, 5′-CATTGAACTTGAAGTACGCGTTAG-3′, reverse, 5′-TTTGCTGTTGTTGGATAGAACG-3′ PSA-ARE: forward, 5′-GCCTGGATCTGAGAGAGATATCATC-3′, reverse, 5′-ACACCTTTTTTTTTCTGGATTGTTG-3′ PLZF-ARE: forward,5′-ACACCATGGCCTGTTGTAAA-3′, reverse, 5′-ACCAAAACAGCAGACCCAAA-3′ NKX3.1-ARE: forward, 5′-CTGGCAAAGAGCATCTAGGG-3′, reverse, 5′-GGCACTTCCTGAGCAAACTT-3′.