IHC analysis was employed to evaluate FGFR protein expression. Antibodies used were anti-FGFR1 (Cell Signaling Technology; 1:500), anti-FGFR2 (R & D Systems; 22 μg/mL), anti-FGFR3 (Cell Signaling Technology; 1:50), and anti-FGFR4 (R & D Systems; 15 μg/mL). PBS was used as negative control. Microscopically, ten high-power (40×) fields from each section were observed randomly and 100 cells were scored. Staining was semi-quantified into four scores based upon the number of positive cells: < 5% (score 0), 5–25% (score 1), 26–50% (score 2), 51–75% (score 3), and > 75% (score 4). Staining intensity followed a four-score classification: no cell stain (score 0), yellow (score 1), tan (score 2) and brown (score 3). Final IHC scores were calculated by addition of intensity and extent scores, and the results were divided as follows: negative (−; scores 0 and 1), weakly positive (+; scores 2 and 3), moderately positive (++; scores 3–5), and strongly positive (+++; scores 6 and 7). Results of staining for FGFR1–4 were organized into low expression (negative and weakly positive) and high expression (moderately and strongly positive) groups [28 (link)].
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