Quantifying ESRP2 Protein Expression

Tissue microarray (TMA) preparation and immunohistochemical staining were performed as previously described for ITGA1 and MYC expression. [32 (link),62 (link)] The same protocol was used herein for ESRP2 using an antigen retrieval procedure based on citrate buffer [62 (link)] and the rabbit anti-ESRP2 antibody 1/200 (GTX123665, GeneTex) overnight followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody 1/100 (GE Healthcare). Images were acquired using a FSX100 Olympus microscope (Olympus, Center Valley, PA, USA) at 20× magnification. ESRP2 staining intensity was graded as a negative/weak (score = 1), moderate (score = 2), strong (score = 3) or very strong (score = 4). ESRP2 staining percentage was graded as 0% to 25% (score = 1) 25% to 50% (score = 2), 50% to 75% (score = 3) or ≥75% (score = 4). Final immunostaining score (intensity X percentage of expression) ranged from 1 to 16. The expression index for each patient was defined as the difference between the expression score for the tumor and normal paired samples: no change (0), increased expression in cancer (positive index = 1), and decreased expression in cancer (negative index = 1).