Ventana discovery automation system

Paraffin-embedded, formalin-fixed, 3-μm tissue sections from human HPC patients were deparaffinized in xylene, and rehydrated by washing with a series of ethanol dilutions and distilled water. Heat-induced epitope retrieval was performed by microwaving the sections in a pH 9.0 target retrieval solution (Dako, Denmark) for CD271 staining or 10 mM citric acid buffer (LSI Medience Corporation, Japan) for involucrin staining. Endogenous peroxidases were blocked with 0.3% H₂O₂. The sections were incubated with anti-human CD271 (1:4,000) for 20 min, or with anti-involucrin (1:1,000) for 90 min, at 37 °C. The CD271-stained sections were incubated for 15 min with mouse LINKER (Dako), followed by incubation with secondary antibody, and development with 3,3′-diaminobenzidine (DAB) Chromogen (EnVision™ Detection SystemsPeroxidase/DAB, Rabbit/Mouse, Dako). Immunostaining with anti-CD271 and anti-involucrin was performed according to the manufacturers’ protocols. Anti-Ki67 staining was performed on a Ventana Discovery automation system (Roche, Switzerland).

Immunohistochemistry (IHC) for Postn was performed using the Ventana Discovery automation system (Roche, Switzerland) according to the manufacturer's protocol. A rabbit polyclonal anti-Periostin antibody (ab14041, 1: 2,000, Abcam, UK) was used according to the manufacturer's instructions on 3-μm sections. Anti-Ki67 (clone 30-9, Roche) staining was also performed using the same system.