Immunohistochemical Analysis of Cellular Markers

Immunohistochemistry was performed as previously described [41 (link)]. Tissue sections were blocked with Background Sniper (BS966, Biocare Medical). Antigen Retrieval was performed in Tris/EDTA pH 9 Buffer for 20 minutes (S2367, Dako). The sections were incubated overnight with the appropriate primary antibody: p^T356RB1 (1:200, 2223-1, Epitomics), RB1 (1:200, #9309, Cell Signaling Technology), p^T202/Y204ERK1/2 (1:100, #9101S, Cell Signaling Technology), N-terminal TP53 (1:200, M7001, DAKO), or Ki-67 (1:800, AC-0009, Epitomics), and pan-cytokeratin (Rabbit 1:400, Z0622, Dako or Mouse, 1:100, M3515, Dako; tumor mask) in Da Vinci Green antibody diluent (PD900, Biocare Medical) at 4°C overnight. Signals were intensified with Envision reagents (DAKO). Pan-cytokeratin primary antibody was probed with an Alexa Fluor 555 dye-labeled secondary antibody (Invitrogen). Primary antibody visualization was accomplished using a Cy-5-tyramide signal amplification system (TSA; AT705A, PerkinElmer). Tissue nuclei were stained using Prolong Gold mounting medium (P36931; Molecular Probes) containing 4,6-diamidino-2-phenylindole (DAPI). HistoRx PM-2000 (HistoRx) with AQUAsition software was used for automated image capture as previously described [41 (link)].