Quantification of pERK1/2 Expression in Xenograft Samples

Immunohistochemistry (IHC) was performed on available paraffin fixed tissue blocks from a xenograft experiment [10 (link)] to detect the expression of pERK1/2. Rehydration of tissue sections was performed in descending concentrations solutions of ethanol (96% to 70%). Antigen was retrieved by heating in a pressure cooker (1 mmol/L Tris-EDTA buffer, 120°C, 5 min). The slices were incubated in blocking solution (Dako, Glostrup, Denmark) to prevent nonspecific binding sites. Next pERK1/2 (1:2000) primary antibody was added to the slices and incubated for 30 min at room temperature. The sections were then washed with washing buffer (Dako) and incubated with EnVision + System horseradish peroxidase-linked secondary antibody (goat anti-rabbit, Dako) at room temperature for 30 min. Positive immunoreactivity was detected using diaminobenzidine + (Dako). Positive and negative IHC controls were included in this study. Percentage and intensity of positively stained epithelial cells (cytoplasmic versus nuclei) was quantified and scored by an expert pathologist (T.T.R.). Statistical analysis was performed using SPSS. Two tailed student t-test was performed by comparing 2 samples assuming that they have equal variances.