The IHC procedure was performed following the method described by Lopes et al (46 (link)). Tumor samples were embedded into paraffin blocks and cut into 3-µm sections. The samples were prepared on silanized glass slides prior to deparaffinization. The sections were rehydrated in an ascending range of alcohol concentrations and incubated with 3% hydrogen peroxide for 30 min. Antigen retrieval was performed by heating at 95°C in buffer for 35 min. The slides were incubated with bovine serum albumin (BSA). The slides were incubated at 4°C overnight with the primary antibodies (Table I ). After being washed with phosphate-buffered saline (PBS) for 15 min, incubation was carried out with Starr Trek Universal HRP Detection kit (Medical Biocare, Concord, CA, USA), consisting of the secondary antibody ‘anti-mouse, rabbit and goat immunoglobulin with biotin’ for 1 h and ‘streptoavidin complex with peroxidase’ for 30 min, followed by washes with PBS for 15 min. Subsequently, 0.5% 3,3′-diaminobenzidine tetrahydrochloride was applied to the slides for 2–5 min at 20–22°C. The slides were counterstained with Harris' hematoxylin for 40 min. Negative controls were obtained by omitting the primary antibody.
The slides were scanned using the Panoramic Digital Slide Scanner (magnification, ×40; 3DHISTECH®) and Panoramic Viewer software (3DHISTECH®) (47 (link)).
The slides were scanned using the Panoramic Digital Slide Scanner (magnification, ×40; 3DHISTECH®) and Panoramic Viewer software (3DHISTECH®) (47 (link)).