*2.5. Immunohistochemistry*

The expression of RUNX1, Ki-67, phospho-pRb (Ser-807/811) proteins in the 409 NSCLC patients was determined using immunohistochemistry of tissue microarrays (TMAs). In brief, the 4-mm–thick TMA tissue sections on glass slide were deparaffinized in xylene and rehydrated in a series of decreasing concentrations of alcohol. Antigens were recovered by putting sections into 10 mmol/L citrate buffer solution (pH 6.0) and by heating in a microwave oven for 10 min. The sections were then incubated overnight at 4 ◦C with a mouse monoclonal antibody to RUNX1 (AML1/RUNX1 Antibody (clone 3A1) IHC-plus™ LS-B5382, LifeSpan BioSciences, Seattle, WA, USA), a polyclonal anti-phospho-pRb (Ser-807/811) antibody (Cell Signaling, Danvers, MA, USA), and a mouse monoclonal anti-Ki-67 (DAKO; clone MIB-1) antibody. Immunoreactivity of the proteins was detected using the Envision-Plus/horseradish peroxidase system (Dako, Carpinteria, CA, USA), and the antibody-bound peroxidase activity was visualized by incubating in 0.05% 3,3 -diaminobenzidine tetrahydrochloride (DAB) for 3 min at room temperature. All sections were counterstained with Mayer's hematoxylin and a negative control was included by excluding the primary antibody each time. Three samples with RUNX1 expression in normal bronchial epithelial cells were used as a positive control for RUNX1 staining, and IHC was performed in duplicate.
