Avidin biotin complexes

For each case, specimens serially sliced to a 4-μm thickness at the central or maximum cross section were selected. After deparaffinization and rehydration, the tissue sections were processed with antigen retrieval by boiling the slides in sodium citrate buffer (10 mmol/L, pH 6.0) for 10 min, followed by immersion in 0.3 % H₂O₂ in methanol for 10 min to quench endogenous peroxidase activity. Non-specific immunoglobulin-binding sites were then blocked with normal serum (Vectastain Elite ABC kit; Vector Laboratories) for 30 min. The sections were incubated with each of the following primary antibodies overnight at 4 °C: rabbit anti-Oct3/4 (1:50; Bioworld) or mouse anti-Nanog (1:10; Abnova). After washing with PBS, the sections were incubated with biotinylated anti-rabbit or mouse secondary antibody for 30 min, which was followed by washing and incubation with avidin-biotin complexes (Vector Laboratories) for 1 h. After a further PBS wash, peroxidase activity was visualized with a 3,3’-diaminobenzidine tetrahydrochloride plus H₂O₂ substrate solution (Vector Laboratories) and counterstained with hematoxylin. The sections were mounted with cover glass and evaluated under a microscope. Immunostaining was defined as positive if nuclear staining was distinctly observed in more than 3 % of the tumor cells.

Following deparaffinization through a series of graded alcohols, endogenous peroxidase activity was quenched in 3% H₂O₂ (in methanol; Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Unspecific antigen-binding sites were blocked using 10% normal serum (in PBS; Abcam, Cambridge, UK), endogenous biotin was inhibited using the Avidin/Biotin Blocking kit (Vector Laboratories, Burlingame, CA, USA) followed by heat-induced epitope retrieval (0.01 M citrate buffer, pH 6.0; 800 W for 6 min). Sections were incubated overnight at 4 °C with antibodies directed against proliferating cell nuclear antigen (PCNA; Abcam), protein inhibitor of activated STAT1 (PIAS1; Abcam) or smooth muscle α-actin (SMA; Sigma). The next day, sections were incubated with secondary antibody (Molecular Probes Inc., Eugene, OR, USA), preformed avidin–biotin complexes (Vector Laboratories) and the peroxidase substrate 3-amino-9-ethylcarbazole (for PIAS and SMA) or 3, 3 -diaminobenzidine (for PCNA) (both Vector Laboratories) until color development. Sections were briefly counterstained with Gill’s hematoxyline (Sigma), mounted in ImmuMount (ThermoScientific, Waltham, MA, USA) and photographed (Olympus BX51 microscope, Olympus Europa SE & Co. KG, Hamburg, Germany).

Paraformaldehyde-fixed paraffin-embedded tissues were cut into 4-μm sections. Paraffin sections were deparaffinized and rehydrated. For antigen retrieval, the slides were autoclaved in 10 mM sodium citrate buffer. Endogenous peroxidase was blocked with 0.345% H₂O₂ for 30 min, and sections were further blocked with 5% BSA in PBS for 30 min. The sections were incubated overnight at 4°C with the following primary antibodies: β-catenin (1:100; BD transduction laboratory, CA, USA) and IGF-1 (1:100; Abcam, MA, USA). Sections were incubated with biotinylated anti-mouse secondary antibody (1:200) (Dako, Glostrup, Denmark) for 1 h at room temperature, followed by incubation with avidin–biotin complexes (Vector Laboratories, CA, USA). Staining was performed with 3,3′-diaminobenzidine (DAB) (Vector Laboratories) as explained in the manufacturer’s protocol. Counterstaining was performed with Mayer’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan). The DAB-stained preparations were visualized with a TE-2000 general optical microscope (Nikon, Tokyo, Japan).

Sections derived from formalin-fixed and paraffin-embedded murine lung tissues were deparaffinized by incubation overnight at 65 °C followed by rehydration in sequential xylene and ethanol rinses. After incubation with hydrogen peroxide, the slides were washed with PBS and then incubated with 0.4% Triton X-100. The sections were incubated with blocking solution (Dako Protein Block, Dako, Glostrup, Denmark) for 30 min at room temperature after washing with PBS. The sections were further incubated with primary antibodies (phosphorylated IGF1R, phosphorylated Src, and cleaved caspase 3 [all from Cell Signaling], diluted at 1:200) overnight at 4 °C, washed with PBS several times, incubated with the corresponding biotinylated secondary antibodies (diluted at 1:500), and then washed with PBS multiple times. After adding avidin-biotin complexes (Vector Laboratories), the sections were visualized using diaminobenzidine (DAB) detection reagent (Enzo Life Sciences, Farmingdale, NY, USA) and mounted with a mounting solution (Vector Laboratories, Burlingame, CA, USA).

Paraffin sections were dewaxed in xylene and hydrated in graded alcohols. Endogenous peroxidase activity was blocked by immersing the slides in 1% hydrogen peroxide in PBS for 15 min. Pretreatment was performed in a steamer using 10 mM citrate buffer (pH 6.0) for 30 min. Sections were incubated overnight with a primary rabbit polyclonal antibody against p40-DeltaNp63 (Abcam, ab166857) diluted at a ratio of 1:100. Sections were washed with PBS and incubated with an appropriate secondary antibody followed by avidin–biotin complexes (Vector Laboratories, Burlingame, CA, PK-6100). The antibody reaction was visualized with 3–3' diaminobenzidine (Sigma, D8001) followed by counterstaining with hematoxylin. Tissue sections were dehydrated in graded alcohols, cleared in xylene, and mounted. For p40 (ΔNp63) IHC, expression was defined based on nuclear labeling.