Immunohistochemistry was performed in normal thyroid and in 211 carcinomas. Briefly, deparaffinized and rehydrated sections were subjected to heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% of hydrogen peroxide and nonspecific binding with Large Volume Ultra V Block reagent (Thermo Scientific/Lab Vision). Sections were then incubated overnight at 4°C with anti-NIS antibody (1:400) clone FP5A (Thermo Scientific/Lab Vision) and in 24 carcinomas with anti-NIS pAb 795 IgG (20 µg/mL) (kindly supplied by Dr Ruby) (36). Additionally, Tyramide Signal Amplification (TSA) Biotin System (Perkin-Elmer) was used for signal amplification in 44 carcinomas, according to manufacturer’s instructions. The detection was performed with a labeled, streptavidin–biotin immunoperoxidase detection system (Thermo Scientific/Lab Vision) followed by 3,3′-diaminobenzidine (Dako) and counterstained with hematoxylin. Graves’ disease sample was used as a positive control and the negative control consisted in omission of the primary antibody.