3 3 diaminobenzidine kit

For bright-field microscopy, free-floating sections were quenched by incubation in a mixture of 3% H₂O₂ and 10% methanol in tris-buffered saline (pH 7.6). Nonspecific binding sites were blocked by incubation in 5% normal serum. Samples were kept overnight at room temperature in a solution containing the primary antibody: rabbit anti–h-αS (1:50,000; ab138501, Abcam), rabbit anti-RFP (1:20,000; 600-401-379, Rockland), mouse anti–Cre recombinase (1:4000; MAB3120, Millipore), rabbit anti–c-fos (1:2000; 2250, Cell Signaling Technology), and rabbit anti-SOD2 (1:5000; ADI-SOD-110, Enzo Life Sciences). Sections were rinsed and incubated in biotinylated secondary antibody solution (1:200; Vector Laboratories). Following treatment with avidin-biotin–horseradish peroxidase complex (ABC Elite kit, Vector Laboratories), color reaction was developed using a 3,3′-diaminobenzidine kit with or without nickel (Vector Laboratories). Sections were mounted on coated slides, coverslipped with Depex (Sigma-Aldrich), and imaged using a Zeiss Observer.Z1 Microscope (Carl Zeiss) equipped with a motorized stage and AxioCam MRm camera (Carl Zeiss). For bright-field density measurements, slides containing MO sections were scanned (AxioScan.Z1, Carl Zeiss). The DMnX was delineated on three equally spaced sections, and integrated density values were obtained using Fiji (ImageJ version 2.1.0/1.53c).

Liver tissues embedded in paraffin were cut into 5 μm-thick sections after fixation in 10% neutral-buffered formalin for >24 h. For staining hepatitis B core antigen (HBcAg)⁺ hepatocytes, liver tissue sections were stained by adding rabbit antibodies against HBcAg (Dako, Carpinteria, CA, USA) followed by biotinylated anti-rabbit IgG and streptavidin-horseradish peroxidase conjugates (Zhongshan Goldenbridge, Beijing, China). The stains were developed using a 3, 3′-diaminobenzidine kit (Vector Laboratories, Burlingame, CA, USA).

Immunohistochemistry (IHC) was performed as described previously [57]. In brief, patient FFPE whole‐tissue sections (4 μm) from radical prostatectomy specimens were deparaffinized; steamed in Antigen Retrieval for 40 min; blocked with Protein Block Serum‐Free (Dako, Agilent Dako, Santa Clara, CA, USA); and then incubated with primary antibodies to ASPN, FAP, THY1, ENG, NT5E, and PDGFRβ (see supplementary material, Table S3) overnight at 4 °C. Primary antibodies were followed by secondary antibodies (Vector Laboratories, Burlingame, CA, USA) and then detected with a 3,3'‐diaminobenzidine kit (Vector Laboratories).

Antigen retrieval for paraffin sections was performed by heating them in citrate buffer (pH 6) for 10 min on full power in a microwave dihydrate buffer (10 mM, pH 6.0) (Sigma-Aldrich) and allowing them to cool and dry. The sections were then washed thrice with PBS (Invitrogen, Renfrew, United Kingdom), followed by permeabilization with 0.1% (v/v) Triton X-100 (Sigma-Aldrich). After washing twice with PBS, non-specific binding was blocked by incubation with 10% (v/v) rabbit, goat, or mouse serum (Vector Laboratories, Burlingame, CA, UnitedStates). The primary antibodies were diluted in PBS with 0.1% (w/v) bovine serum albumin (Sigma-Aldrich) and 0.01% (w/v) sodium azide (Sigma-Aldrich). Sections were incubated with primary antibodies overnight at 4°C (Table 1). The sections were then washed thrice with PBS and incubated for 1 h with biotinylated goat anti-rabbit secondary antibody diluted 1:250 in PBS (Vector Laboratories). An ABC kit was applied for 30 min and a 3,3′-diaminobenzidine kit for 2–10 min to stain the nuclei (Vector Laboratories).

Liver tissues were fixed in 12% neutral buffered formalin to observe liver slides. For PCNA staining, liver slides were stained with a 1:100 dilution of an anti-PCNA (ZSGB-BIO, Beijing, China) primary antibody overnight, and this was followed by a polymer HRP detection system (ZSGB-BIO, Beijing, China). Brown stains were developed using a 3,3-diaminobenzidine kit (Vector Laboratories, Burlingame, CA).