3 3 diaminobenzidine tetrahydrochloride dab

Deparaffinised sections were pre-treated for antigen retrieval in a 1,500-W microwave oven for SV40 TAg and by incubation with proteinase K solution (Roche, Mannheim, Germany) for BKPyV VP1. The sections were pre-incubated with 5% non-fat milk for 60 min to eliminate non-specific binding. Then, the sections were incubated with the first antibody (anti-SV40 TAg antibody [1:400, DP02-200UG, mouse monoclonal, Calbiochem, San Diego, CA, USA] and anti-BKV VP1 antibody [1:500, MAB3204-M22, mouse monoclonal, Abnova, Taipei, Taiwan]) which were diluted in phosphate buffered saline (PBS)/1% bovine serum albumin (BSA) overnight at 4°C. After extensive washing, the sections were incubated with peroxidase-labelled secondary antibody (100 mg/mL; Nichirei Corp., Tokyo, Japan), stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Nichirei). Counterstaining was performed with haematoxylin, and sections were coated with glycerol and covered. For negative controls, sections were treated with normal murine IgG (Santa Cruz Biotechnology, Dallas, TX, USA) as the primary antibody.