Ventana benchmark xt automated system

We selected formalin-fixed paraffin-embedded (FFPE) tissue samples from rectal cancer tissues and used them for tissue microarray (TMA) construction. Of the 102 patients with rectal cancer, tissue specimens from both before CRT (biopsy specimen) and after CRT (surgery specimen) were selected for 71 patients. Only biopsy specimens were selected from 31 patients with no or few cells remaining in the surgical specimens. Representative tumor areas were identified by two pathologists (H.O. and H.W.L.) in hematoxylin and eosin-stained tissue sections. After the pathologists’ review, TMAs were assembled from triplicate 5 mm cores of the FFPE tumor samples. Immunohistochemical staining was performed on the TMA sections with an antibody against PLK4 (12952-1-AP, 1:400, ProteinTech Group, Inc., Chicago, IL, USA) using the Ventana BenchMark XT Automated System following the manufacturer’s protocol (Ventana Medical Systems, Tucson, AZ, USA).

Immunohistochemical studies were performed for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD20, CD30, CD56, Bcl-2, Bcl-6, C-MYC, MUM-1, PD-1, ICOS, ALK, TdT, TCRβF1, TCRγ, granzyme B, pHH3, and Ki-67 on available formalin-fixed paraffin-embedded tissue (FFPE) sections. Information on the antibodies and staining methods are summarized in Supplementary Table S1 (Supplementary Digital Content 2, http://links.lww.com/PAS/B299). Epstein-Barr virus (EBV) in situ hybridization was performed using the Bond Ready-to-Use ISH EBV-encoded RNA (EBER) probe (Leica Biosystems, Newcastle, UK) or INFORM EBER Probe (Ventana Medical Systems, Tucson, AZ), in conjunction with the Bond-Max autostainer (Leica Microsystems) and Ventana Benchmark XT automated system (Ventana Medical Systems), respectively, according to the manufacturer’s protocol.