Immunohistochemistry (IHC)

Immunohistochemical analyses were performed using primary antibodies against PFN2 (1:100, PK07518; Abmart) and Ki-67 (1:100,9027 s; Cell Signaling Technology). Tissue specimens were deparaffinized, rehydrated, and treated with boiling citrate buffer for 6 min for antigen extraction. Sections were enclosed in a blocking reagent (Millipore) for 10 min, incubated overnight with primary antibodies at 4 °C, washed with tris buffered saline with 0.1% Tween 20 (TBST), and then incubated with secondary antibodies (Millipore) for 45 min. An HRP/DAB kit (Millipore) was used for staining. IHC images were obtained under a microscope at 10x and 20x objectives. IHC staining was scored by the percentage of positive area and intensity as follows: 0, no staining; 1, <10% positive, moderate, or strong intensity; 2, 10–50% positive, moderate, or strong intensity; 3, >50% positive, moderate intensity; and 4, >50% positive, strong intensity.