Optimization of tissue processing for immunohistochemistry for the detection of human glypican-3

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 24h 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.