Ventana benchmark xt automated stainer

Formalin-fixed, paraffin-embedded tissues were cut at a thickness of 4 to 5 μm. Dual- and single-marker staining were performed on sections according to a standard immunostaining protocol using a Ventana Benchmark XT Automated Stainer, following the manufacturer’s instructions. Briefly, the sections were heated for 1 hour at 58°C, deparaffinized, and dehydrated in a graded alcohol series. Antigen retrieval was performed using Cell Conditioning 1 (Ventana) Tris/Borate/EDTA for 60 minutes with a Ventana Benchmark XT Automated Stainer followed by dual-marker immunostaining for p40/napsin A and CK5/6/TTF1, according to the manufacturer’s instructions. The sections were blocked with hydrogen peroxide and normal goat serum and then incubated with a mixture of anti-p40 and anti-napsin A or anti-CK5/6 and anti-TTF1 antibodies at ratios of 1:1 for 30 minutes at 37°C. The antibody concentrations were about 1.0 µg/mL, respectively. The color was developed with 3,3’-diaminobenzidine (DAB), and the sections were then counterstained with hematoxylin. Positive controls were single-stained for p40, napsin A, CK5/6, or TTF-1. Single-marker immunostaining for p40, napsin A, CK5/6, and TTF1 was also performed synchronously using an UltraView DAB Detection Kit and Ventana Benchmark XT Automated Stainer, according to the manufacturer’s instructions.

Anti-p40 (clone ZR8), anti-TTF1 (clone MX011), anti-CK5/6 (clone D5/16B4), and anti-napsin A (clone MX015) antibodies and the antibody diluent (ABD-0030, IVD) were all procured from Maixin Biotechnology Co., Ltd. (Fuzhou, China). The antibodies were used for IHC at dilutions of 1:100 in conjunction with an UltraView DAB Detection Kit and Ventana Benchmark XT Automated Stainer (Ventana Medical Systems, Shanghai, China).