Sc 186

Immunohistochemical staining was performed on representative tissue sections cut from formalin-fixed, paraffin-embedded tissues at 3-μm thickness as our previous study [24 (link)] with a few modifications. Slides were deparaffinized with xylene, rehydrated with ethanol, heated by microwave for retrieval of antigen epitopes in a 10 mM citrate buffer (pH 6) for 7 min. Endogenous peroxidase was quenched by 3% H₂O₂. Slides were washed with Tris buffered saline for 15 min and then incubated with a primary monoclonal antibody against EMP2 (1:20; HPA014711, Sigma-Aldrich), CREB1 (1:40, sc-186, Santa Cruz) and pCREB1(S133) (1:50, sc-7978, Santa Cruz), for 1 h, followed by antibody detection using a ChemMate EnVision™ kit (K5001; DAKO, Glostrup). Two pathologists (CF Li and HY Huang) blinded to clinicopathological information and patient outcomes, independently interpreted the immunostainings. The immunointensity was scored based on the extent of moderately to strongly-stained tumor cells exhibiting combined membranous and cytosolic (EMP2) or nuclear [CREB and pCREB(S133)] staining, and labeled as 0+, < 5%; 1+, ≥ 5%, but < 25%; 2+, ≥ 25%, but < 50%; 3+, ≥ 50%, but < 75%; and 4+, ≥ 75%, respectively. A specimen showing EMP2 staining less than 1+ was regarded as loss of EMP2 expression. For CREB1 and pCREB1(S133), 4+ staining were regarded as high expression.

IHC was performed on paraffin-embedded tissue sections. After deparaffinization, rehydration, and antigen retrieval with citrate buffer (Invitrogen), slides were permeabilized with 0.5% triton X-100. Slides were then blocked using a ready-to-use IHC kit (BioVision) as described by the manufacturer. Slides were then incubated with primary antibodies: anti-CXCR7 (1:50; R&D, MAB42273), mouse IgG control (1:100; R&D, MAB002), anti-phospho-Aurora A (Thr288) (1:1000; CST, 3079), mouse anti-SYP (1:500; Santa Cruz, sc-17750), and anti-AR (1:200; Santa Cruz, SC-186) overnight at 4°C in a humidity chamber. Slides were then washed with 1 × TBS 3 times for 5 minutes each time. For secondary antibodies, slides were incubated with IBSC-1-step HRP-anti-mouse, rat, and rabbit polymer provided in the kit. Slides were washed again with TBS (3 times for 5 minutes each time), then incubated with 3,3′- Diaminobenzidine (DAB) substrate for 1–5 minutes at room temperature. Slides were then counterstained with hematoxylin for 15 seconds, washed with running tap water, dehydrated in ethanol, cleared with xylene, and mounted with Permount (Fisher Chemical). Slides were visualized and imaged with an Olympus BX41 microscope bound to with Olympus UTV 0.5XC3 camera.