Ab151562

At least three 4-micrometer sections were taken from different mouse prostates for each of the genotypes surveyed. Sections were used for H&E staining and for PRR and PACE IHC. Slides were heated for 20 minutes at 60˚C, deparaffinized in xylenes and rehydrated in ethanol gradient. Antigen retrieval was performed using EDTA pH 8.0 (PRR) buffer and sodium citrate pH 6.0 (PACE4). PACE4 slides were further autoclaved in 10 mM citrate buffer pH 6 for 45 min (16 psi, 121 ˚C). To deactivate endogenous peroxidase, 3% H₂O₂ was used, and slides were blocked using a protein blocking reagent (Dako) for PRR and an IHC blocking buffer (Pierce) for PACE4, at room temperature. Tissue sections were incubated with primary antibodies diluted in 5% BSA in TBST overnight at 4˚C (Abcam, ab64957, 1:600; PACE4: Abcam, ab151562, 1:400) and then with biotin-conjugated secondary antibodies (Santa-Cruz) for PRR and a secondary HRP-conjugated anti-Rabbit antibody (Biorad) for PACE4. Staining was developed using DAB (Sigma-Aldrich) containing 0.015% H2O2. Counterstaining was performed with hematoxylin.

Protein concentrations were initially measured using the BCA method (Thermo Fisher Scientific). Thereafter, total protein (25 μg/well) was fractionated on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred overnight to nitrocellulose membranes (Millipore, Billerica, MA, USA), which were then incubated sequentially with 5% nonfat dry milk at room temperature for 1 hr, primary antibodies at 4°C overnight, and then secondary antibody anti-mouse IgG (1 : 1,000; Beyotime, Shanghai, P.R. China) at 37°C for 1 hr. An enhanced chemiluminescence system (Tanon, Shanghai, P.R. China) was used to determine the protein levels. The primary antibodies used included the following: PCSK6 (Ab151562, Abcam, UK), CD63 (25682-1-AP, Proteintech, USA), CD81 (66866-1-Ig, Proteintech, USA), and GAPDH (60004-1-1 G, Proteintech, USA).