Sc 5958

IHC staining for the detection of TFE3 was performed using a goat polyclonal antibody for TFE3 (sc-5958, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the Envision detection kit (Dako, Carpinteria, CA, USA) in 76 suspected Xp11.2 RCC. The detailed procedures of immunostaining was carried out as mentioned in previous study29 (link).
All slides were examined and scored by two pathologists, who were blinded to all clinical data, in an open discussion. The interpretation of immunoreactivity for TFE3 was evaluated as previously reported20 (link)30 (link). Tumors scored as positive for TFE3 demonstrated nuclear immunoreactivity that was readily apparent at low-power magnification (×4 objective). These cases were subdivided into moderately (2+) and strongly (3+) positive on the basis of the intensity of labeling. Weak/equivocal nuclear immunoreactivity (1+) for TFE3, demonstrating nuclear immunoreactivity was subtle at low magnification and typically required higher magnification to be appreciated, was considered as negative. Cytoplasmic immunoreactivity was ignored because native TFE3 and its fusion proteins are known to localize to the nucleus31 (link).

Immunohistochemistry of TFE3 were performed on the four-μm-thick formalin-fixed paraffin-embedded (FFPE) sections of all the RCC cases. IHC staining of the sections was carried out using anti-TFE3 antibody (SC-5958,1:300; Santa Cruz, CA). Further, IHC assay of the TFE3 was conducted though labeled streptavidin-biotin method followed by overnight incubation (22 (link)). The Xp11.2 translocation RCC and IgG were used as positive and negative controls for the IHC assay, respectively. Only nuclear TFE3 staining was considered as the positive result. The final result was analyzed by two independent observers and the inconsistent result was resolved by an experienced pathologist. To perform a semi-quantitative assessment, the results of this study were analyzed in reference to the previously reported intensity and degree of nuclear staining (22 (link), 23 (link)). The IHC score was calculated by multiplying the percentage of positive cells (0–100) by the staining intensity (0=no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining). For the result of immunostaining, a score of between 0 and 25 was considered negative (–), a score of between 26 and 100 was considered weak (+), a score of between 101 and 200 was considered moderate (++), and a score of between 201 and 300 was considered strong (+++).