Ap conjugated anti α smooth muscle actin antibody

Prostate lobes were separated by microdissection, and embedded in paraffin for sectioning ^{24 (link)}. After antigen retrieval in hot citrate buffer, sections were blocked in 5% of normal horse serum and 1% of normal goat serum, and subjected to immunohistochemistry staining using the ABC Elite Kit and the DAB Kit (Vector) according to the manufacturers’ recommendations. The following antibodies were used: Ki-67 (ab15580, 1:600) from Abcam; ATF3 (Santa Cruz sc-188, 1:200), AR (sc-816, 1:200), and p63 (sc-8430, 1:200) from Santa Cruz; p-AKT S473 (#4060, 1:200), p-AKT T308 (#2965,1:200), AKT (#4691, 1:200), p-S6 (#2211, 1:400), S6 (#2217, 1:200), cleaved caspase-3 (#9661, 1:300), and Pten (#9188, 1:100)from Cell Signaling; MMP2 (NB200-193, 1:200) from Novus; and MMP-9 (ab137867, 1:1000) from Abcam. For α-smooth muscle actin (α-SMA) staining, sections were incubated with alkaline phosphatase (AP)-conjugated anti-α-smooth muscle actin antibody (Sigma, 1:600) followed by detection of AP activity using the SIGMAFAST Fast Red TR/Naphthol AS-MX tablets (F4523, Sigma) according to the supplier’s protocol. For quantifying IHC staining intensity, random microscopic fields were captured and digitized by a CCD camera (Olympus). Signal intensity was determined using the Image-Pro Plus software and presented as integrated optical density (IOD).

These were carried out essential as described previously^{13 (link)}. In brief, prostate sections were treated with a hot citrate buffer, and subjected to IHC staining using the ABC Elite Kit and the DAB Kit (Vector laboratories, Burlingame, CA, USA) according to the manufacturers’ protocols. PCNA (sc-56, 1:1000), ATF3 (sc-188, 1:200), AR (sc-816, 1:200), and p63 (sc-8430, 1:200) antibodies were purchased from Santa Cruz (Dallas, TX, USA). α-smooth muscle actin (α-SMA) staining were performed using an alkaline phosphatase (AP)-conjugated anti-α-smooth muscle actin antibody (Sigma, 1:600) and SIGMAFAST Fast Red TR/Naphthol AS-MX tablets (F4523, Sigma-Aldrich, St Louis, MO, USA) according to the supplier’s protocol. For CK5/CK8 double staining, prostate sections were incubated with CK5 (PRB-160P, 1:500) and CK8 (MMS-162P, 1:500) antibodies (Biolegend, San Diego, CA, USA), followed by incubation with Alexa Fluor 594-conjugated anti-rabbit IgG (A-24923) and Alexa Fluor 488-conjugated anti-mouse IgG (A-21131, Life Technologies).