Envision reagent

Immunohistological experiments using surgically obtained tumor tissues from the patients were approved by the research ethics committee of the Niigata University (authorization number; #2015‐2334, 2016‐0089), and informed consent was obtained from all patients to perform the analyses. Immunohistochemistry was implemented using paraffin‐embedded section. Deparaffinization of paraffin‐embedded section was accompanied by antigen retrieval by using Pascal pressure chamber (Dako, USA) in sodium citrate buffer. Antigen‐retrieved sections were incubated with anti‐human Crb3a monoclonal (1 μg/mL, generated in our study) and/or anti‐FGFR1 (1:100 dilution, #sc‐121, Santa Cruz Biotechnology, USA) in PBS including 1.0% bovine serum albumin (#017‐25771, Wako Pure Chemical Industries), and stained using EnVision+ reagent (#K400011, Dako). DAB‐stained sections were counterstained with Mayer's hematoxylin (#30004, Muto Pure Chemicals, Japan). Fluorescent dye‐labeled secondary antibodies were employed for double staining.

Five-micrometre-thick paraffin-embedded sections from tumour samples were deparaffinised, rehydrated, immersed in 10 mM l⁻¹ sodium citrate buffer, pH 6 and microwave heated for 4–5 min cycles at 750 W. After rinsing, endogenous peroxidase activity was inactivated with 3% H₂O₂. Sequential sections were then incubated overnight at 4 °C with specific anti-SERPINB3 monoclonal antibody (1 : 20, SCCA-1 clone 8H11, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-TGF-β1 monoclonal antibody (1 : 300, TGFβ1 clone 3C11, Santa Cruz Biotechnology) and anti-β-catenin monoclonal antibody (1 : 500, β-catenin clone N1N2-2, GeneTex, Irvine, CA, USA). After incubation with the primary antibody, immunostaining was performed using the ready-to-use, peroxidase-conjugated EnVision reagent (DakoCytomation, Carpinteria, CA, USA). For quantitative analysis, 10 images of representative fields of 9 HCC samples (5 with low SERPINB3 mRNA expression and 4 with high SERPINB3 mRNA expression) were captured by Leica Qwin Plus v3 software (Cambridge, UK), under a CCD camera connected to a Leica microscope (Microsystem Imaging Solutions Ltd, Cambridge, UK). The average sum of intensities and stained-area percentage of each sample was calculated using ImageJ software (http://rsb.info.nih.gov/ij). Negative controls, obtained by replacing primary antibodies with PBS, were always run in parallel.

Tissues were fixed in 4% paraformaldehyde and paraffin embedded. For immunohistochemistry (IHC), specimens were incubated with antibodies against alpha melanocyte-stimulating hormone (α-MSH), calcitonin, E2F1/2/3, prolactin (PRL), luteinizing hormone/follicle-stimulating hormone alpha subunit (LH/α-FSH), thyroid-stimulating hormone (TSH) and growth hormone (GH; antibody sources, Supporting Information Table S1), followed by incubation with Envision reagent (DAKO, Glostrup, Denmark). The HRP substrates and alkaline phosphatase (AP) substrates were used in the chromogenic reactions. Labeled specimens were scored for the number of positive cells per three high-powered fields per sample.

Western blotting was performed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Briefly, 20‐μg homogenates of sural nerve were resuspended in a reduced sample buffer, electrophoresed on a 10% Tris gel with Tris running buffer, blotted to a polyvinylidene difluoride membrane, and sequentially probed with polyclonal rabbit antihuman CD10 antibody (Abcam). The membrane was subsequently incubated with a horseradish peroxidase–labeled polymer‐conjugated antimouse antibody reagent (EnVision+ reagent; Dako, Tokyo, Japan). 3‐3′‐diaminobenzidine was used for chromogenic visualization.