Dab chromogen and substrate mixture

Paraffin embedded tissue sections derived from tumor bearing mice were deparaffinized with xylene, rehydration with graded concentrations of ethanol, and then practice antigen recovery with 10 mM citrate buffer (pH 6.0) via Heat-Induced Epitope Retrieval (HIER) method, following by blocking endogenous peroxidase with 3% H₂O₂ in TBS for 15 min. Samples were blocked with Ultra V block (Thermo Scientific, Waltham, MA, U.S.A.) and incubated with specific antibodies at a 1:100 dilution for overnight at 4°C. The tissue sections were rinsed with TBST three times per five minutes and then incubated with HRP conjugated antibodies (Santa Cruz Biotechnology, Inc.). Excess antibodies were removed by rinsing with TBST three times per five minutes and tissue specimens were then reacted with DAB chromogen and substrate mixture (Thermo Scientific) for appropriate timing. Immunostaining was visualized after couterstaining with hematoxylin. All of antibodies used in present study were listed in Supplementary Material file.

Paraffin embedded tissue sections were derived from commercial human PCa tissue array (SUP-CA array from SuperBioChips, Seoul, Korea and PR956a array from US Biomax, Rockville, MD, USA) or tumor bearing mice. Paraffin embedded tissue sections were deparaffinized with xylene, rehydration with graded concentrations of ethanol, and then practice antigen recovery with 10 mM citrate buffer (pH 6.0) via Heat-Induced Epitope Retrieval method, and blocked endogenous peroxidase with 3% H₂O₂ in TBS for 20 min. Samples were blocked with Ultra V block (Thermo Fisher Scientific) and incubated with primary antibody at a 1:100 dilution for overnight at 4 °C. The tissue sections were rinsed with TBST three times per 5 min, and then incubated with HRP conjugated antibodies (N-Histofine, NICHIREI Biosciences, Tokyo, Japan). Excess antibodies were removed through being rinsed with TBST three times per 5 min, and then tissue specimens were reacted with DAB chromogen and substrate mixture (Thermo Fisher Scientific) for appropriate timing. Immunostaining was visualized after counterstaining with Hematoxylin. Scoring of immunohistochemistry of commercial tissues arrays were evaluated independently by two pathologists. All of antibodies used are listed in Supplementary Table 2.