Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding

Yasuyoshi Mizutani, Kazuya Shiogama, Ken Ichi Inada, Toshiyuki Takeuchi, Atsuko Niimi, Motoshi Suzuki, Yutaka Tsutsumi

Research output: Contribution to journalArticlepeer-review

Abstract

The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemo-cyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activ-ity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.

Original languageEnglish
Pages (from-to)129-148
Number of pages20
JournalActa Histochemica et Cytochemica
Volume55
Issue number5
DOIs
Publication statusPublished - 2022

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Biochemistry
  • Physiology
  • Histology
  • Cell Biology

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