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Fluorescein isothiocyanate (FITC)-labeled antibodies are widely used as primary antibodies in the tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies. However, the effects of FITC-labeling on the characteristics of an antibody are poorly understood. The present study was performed to examine the effect of FITC-labeling on the binding affinity and immunohistochemical staining profile of an antibody, using several antibodies with different FITC-labeling indices. The results showed that the FITC-labeling index in antibody was negatively correlated with the binding affinity for its target antigen. Immunohistochemically, an antibody with a higher labeling index had a tendency to be more sensitive, but was also more likely to yield non-specific staining. Based on these findings, we recommend that a FITC-labeled antibody used as a primary antibody in a TCR study should be carefully selected from several differently labeled antibodies to minimize the decrease in the binding affinity and achieve the appropriate sensitivity and interpretation of the immunohistochemistry.

Summary Glypican-3 (GPC3) is frequently upregulated in hepatocellular carcinoma (HCC) and data on the expression profile in HCC might be useful for therapeutic decision-making and prognostic prediction. This study was performed using HepG2 xenograft tissues to optimize the tissue processing method for GPC3 immunohistochemistry. The optimization was conducted in terms of using GPC3 immunohistochemistry for biological study of GPC3 (Experiment 1) and as a diagnostic tool (Experiment 2). In Experiment 1, GPC3 immunoreactivity (IR) and tissue architecture were compared among differently fixed and embedded specimens. In Experiment 2, using conventional formalin-fixed paraffin-embedded (FFPE) procedures, the effects of different fixation times and antigen retrieval treatments were assessed. In Experiment 1, the periodate-lysine-paraformaldehyde (PLP)-fixed and AMeX method-embedded (PLP-AMeX) specimen showed superior immunoreactivity and excellent tissue architecture preservation. In contrast, the other specimens, especially frozen specimens, resulted in poor IR. In Experiment 2, specimens fixed for 24 h showed better IR than those fixed for 7 days and the most remarkable improvement in IR was achieved after protease treatment. These findings indicate that with GPC3 immunohistochemistry for biological studies, the PLP-AMeX specimen is preferable. For diagnostics using FFPE specimens, the fixation time should not be too long and protease should be used for the antigen retrieval treatment.