Diaminobenzidine dab

The 4 μm thick paraffin-embedded formalin-fixed tissue sections were de-paraffinized, and heat-based antigen retrieval was performed in 0.1 mol/L citrate buffer (pH 6.0). Endogeneous peroxidase activity was inhibited using hydrogen peroxide. The primary antibody for P-gp (ab98322) was purchased from Abcam (Cambridge, MA). The detection system was the streptavidin-biotin-peroxidase complex technique (ABC kit; Vector Laboratories, Burlingame, CA) with diaminobenzidine (DAB; Nichirei Bioscience, Tokyo, Japan) as the chromogen. In situ hybridization (ISH) of Epstein–Barr virus-encoded small RNA (EBER) was performed for detection of EBV in tissue sections by EBV (EBER) PNA Probe/Fluorescein (DAKO, Carpinteria, CA) and second antibody for Fluorescein (Dako, Glostrup, Denmark). For double staining for EBER and P-gp, anti-P-gp staining was performed with nickel DAB as chromogen (Vector Laboratories, Burlingame, CA), followed by the ISH.

FFPE tissue sections (4-μm thick) were used for immunohistochemistry. Deparaffinization was followed by heat-based antigen retrieval, endogenous peroxidase blockade with 3% hydrogen peroxide, and blocking with normal sera. The primary antibodies used were as follows: MCM2, mouse monoclonal, 1:2000 (BD Biosciences, San Jose, CA, USA); CD133, mouse monoclonal, 1:100 (Miltenyi Biotec, Auburn, CA, USA); ALDH-1, rabbit monoclonal, 1:1000 (Abcam, Cambridge, UK); FLAG, mouse monoclonal, 1:250 (InvivoGen, San Diego, CA, USA); and GFP, mouse monoclonal, 1:100 (Abcam). The primary antibodies were incubated overnight at 4°C. Primary antibodies were detected using an ABC Kit (Vector Laboratories, Burlingame, CA, USA) or EnVision⁺ System-HRP (Dako, Glostrup, Denmark) with diaminobenzidine (DAB; Nichirei Bioscience, Japan) or the HISTOFINE simple stain AP series (Nichirei Bioscience) with Vector Blue (Vector Laboratories). A TUNEL assay was also performed using the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Tokyo, Japan) with DAB. For double immunostaining, samples were heat treated and blocked between each step.