Immunohistochemistry Protocols for Protein Expression

For immunohistochemistry, TMA was constructed, as described by Pires et al. (19 (link)). All samples were fixed in 10% formalin and embedded in paraffin. Serial TMA sections were cut, mounted on glass slides and dried at 56°C before dewaxing in xylene and rehydration in alcohol. All sections were subjected to heat-induced epitope retrieval in citrate buffer followed by inhibition of endogenous peroxidase (peroxidase block, RE7101, Novocastra, UK). Incubation of primary antibodies against EGFR (D38B1, 1:25, Cell Signaling Technology), ERCC1 (1:100; 8F1 clone, Thermo Fisher Scientific), or p53 (1:100; G59–12 clone, BD Pharmingen) was performed for 1 h. Slides were incubated with NovoLink™ polymer (RE7112, Novocastra), further developed with diaminobenzidine chromogen (RE7105, Novocastra), and finally stained with Mayer hematoxylin, dehydrated and mounted with Canadian balsam. EGFR, ERCC1 and p53 expressions were evaluated semi-quantitatively as positive cells after counting 300–500 tumor cells, being scored as negative (no staining), 1+ (<25% of positive cells), 2+ (25–75% of positive cells) or 3+ (>75% cells staining positively). Negative controls were obtained by omitting the primary antibody.