{"title":"酶标抗原法:影响福尔马林固定和石蜡包埋过程中特异性抗体抗原结合活性下降的因素。","authors":"Yasuyoshi Mizutani, Kazuya Shiogama, Ken-Ichi Inada, Toshiyuki Takeuchi, Atsuko Niimi, Motoshi Suzuki, Yutaka Tsutsumi","doi":"10.1267/ahc.22-00023","DOIUrl":null,"url":null,"abstract":"<p><p>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 hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) 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.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":1.6000,"publicationDate":"2022-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/03/3b/ahc-055-129.PMC9631987.pdf","citationCount":"0","resultStr":"{\"title\":\"Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding.\",\"authors\":\"Yasuyoshi Mizutani, Kazuya Shiogama, Ken-Ichi Inada, Toshiyuki Takeuchi, Atsuko Niimi, Motoshi Suzuki, Yutaka Tsutsumi\",\"doi\":\"10.1267/ahc.22-00023\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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 hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) 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.</p>\",\"PeriodicalId\":6888,\"journal\":{\"name\":\"Acta Histochemica Et Cytochemica\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2022-10-28\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/03/3b/ahc-055-129.PMC9631987.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Acta Histochemica Et Cytochemica\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1267/ahc.22-00023\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2022/10/25 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Acta Histochemica Et Cytochemica","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1267/ahc.22-00023","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2022/10/25 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding.
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 hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) 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.
期刊介绍:
Acta Histochemica et Cytochemica is the official online journal of the Japan Society of Histochemistry and Cytochemistry. It is intended primarily for rapid publication of concise, original articles in the fields of histochemistry and cytochemistry. Manuscripts oriented towards methodological subjects that contain significant technical advances in these fields are also welcome. Manuscripts in English are accepted from investigators in any country, whether or not they are members of the Japan Society of Histochemistry and Cytochemistry. Manuscripts should be original work that has not been previously published and is not being considered for publication elsewhere, with the exception of abstracts. Manuscripts with essentially the same content as a paper that has been published or accepted, or is under consideration for publication, will not be considered. All submitted papers will be peer-reviewed by at least two referees selected by an appropriate Associate Editor. Acceptance is based on scientific significance, originality, and clarity. When required, a revised manuscript should be submitted within 3 months, otherwise it will be considered to be a new submission. The Editor-in-Chief will make all final decisions regarding acceptance.