title: Optimization of tissue processing for immunohistochemistry for the detection of human glypican-3.
abstract: 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.
authors.affiliation: Safety Assessment Department, Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. takaihrt@chugai-pharm.co.jp
authors.forename: Hirotake
authors.initials: H
authors.lastname: Takai
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Kato
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishiguro
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Kinoshita
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Karasawa
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Otani
authors.forename: Masamichi
authors.initials: M
authors.lastname: Sugimoto
authors.forename: Masami
authors.initials: M
authors.lastname: Suzuki
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kataoka
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D005404:Fixatives; D005557:Formaldehyde; D053673:Glypicans / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008239:Lysine; D008297:Male; D051379:Mice; D016513:Mice, SCID; D016612:Paraffin Embedding / Q000379:methods*; D010504:Periodic Acid; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*; D019737:Transplants
chemical_list: D000911:Antibodies, Monoclonal; D005404:Fixatives; D053673:Glypicans; C046311:periodate-lysine-paraformaldehyde; D010504:Periodic Acid; D005557:Formaldehyde; D008239:Lysine