Ma182041

Four-micrometer BPH sections and PCa tissue sections were cut and placed onto positively charged slides (MIC3040, Scientific Laboratory Supplies) and polyethylene naphthalate (PEN) membrane glass slides (LCM0522, Applied Biosystems). For every sample, four unstained sections were prepared. The immunostaining was performed using the primary antibodies DKK3 1:300 dilution (Ab187532 rabbit monoclonal, Abcam), ECM-1 1:200 dilution (11521-1-AP rabbit monoclonal, Proteintech), TGFBI 1:200 dilution (Ab170874 rabbit monoclonal, Abcam), and PCK-1 1:200 dilution (MA182041 mouse monoclonal, Invitrogen). EnVision+ System-HRP-labeled polymer anti-rabbit (K400311, DAKO) and anti-mouse (K400111, DAKO) were used as secondary antibodies.

Fourteen patients in total, two slides per patient were stained for DKK3 (Ab187532, Abcam) and pan-cytokeratin (MA182041, Invitrogen).
Three randomized pictures were taken using a Leica DM750 microscope and scored based on the staining intensity as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining).

NE-treated cells were fixed for 15 min using 4% paraformaldehyde and then blocked for 30 min using 5% donkey serum containing 0.3% Triton X-100. The cells were then incubated with the primary antibodies against CHGB (1 : 100; rabbit; PA5-52605, ThermoFisher Scientific) or SYP (1 : 100; rabbit; MA5-14532, ThermoFisher Scientific) for 3 and 1 h, respectively, followed by incubation with anti-rabbit Alexa Fluor® 488 Conjugate (1 : 100, A11034, ThermoFisher Scientific) secondary antibody for 1 h at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.
For Adrβ2 and cytokeratin expression in tumor and adjacent normal tissues, the tissues were incubated with primary antibodies against Adrβ2 (1 : 100; rabbit; PA5-14117, ThermoFisher Scientific) and cytokeratin (1 : 100; mouse; MA1-82041; ThermoFisher Scientific) for 1 h at room temperature, followed by incubation with a cocktail of anti-rabbit Alexa Fluor® 488 Conjugate (1 : 100, A11034, ThermoFisher Scientific) and anti-mouse Alexa Fluor® 546 (1 : 100, A21045, ThermoFisher Scientific) for 1 h at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.