Immunohistochemical Quantification of Nucleolin

Immunohistochemistry analysis was performed as previously described with minor modifications [52 (link)]. Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at 5 µm thickness, deparaffinized, and rehydrated. Antigen were unmasked by heat retrieval with pH 9 EDTA buffer for 15 min and endogenous peroxidase activity was inactivated with a 3% hydrogen peroxide solution for 10 min. Tissues were then immuno-stained overnight at 4 °C with anti-nucleolin antibody (sc-8031, Santa-Cruz Biotechnology, Heidelberg, Germany, 1:50) and diluted in antibody diluent (Zytomed, Berlin, Germany). Immuno-complexes were revealed using the anti-mouse Polink HRP mouse kit and the DAB substrate (Diagomics, Blagnac, France) with an incubation of 30 min at room temperature. Tissues were then stained with hematoxylin and dehydrated. Slides were mounted using Eukitt medium. Protein expression was scored as null (0), weak (1), moderate (2), and strong (3), and the percentage of tumor cells stained was noted. The multiplication of the score and the percentage of stained tumor cells gave the quick score (between 0 and 300). Analysis was performed separately by one genitourinary pathologist (MN) and by two scientific investigators (DD and VF).