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(American Journal of Pathology. 2000;157:1829-1838.)
© 2000 American Society for Investigative Pathology

Technical Advance

Co-Localization of Multiple Antigens and Specific DNA

A Novel Method Using Methyl Methacrylate-Embedded Semithin Serial Sections and Catalyzed Reporter Deposition

Marcus Mueller*, Karin Wacker*, William F. Hickey{dagger}, Erich B. Ringelstein* and Reinhard Kiefer*

From the Department of Neurology,*
Westfälische Wilhelms-Universität, Münster, Germany; and the Department of Pathology,{dagger}
Dartmouth Medical School, Lebanon, New Hampshire

Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the process necessary to detect the target structure of one technique may negatively affect the target of the other. Morphological impairment may also limit the application of the two techniques on sensitive tissue. To overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on semithin sections of methyl methacrylate-embedded tissue. Microwave-stimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for both techniques, yielding high sensitivity and excellent morphological preservation compared to conventional paraffin sections. Co-localization of in situ hybridization and immunohistochemistry signals with high morphological resolution was achieved on single sections as well as on adjacent multiple serial sections, using computerized image processing. The latter allowed for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level. The method was successfully applied to radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into TK-tsa transgenic Lewis rats, in an attempt to trace and characterize TK-tsa transgenic cells. It also proved useful in the co-localization of multiple antigens in peripheral nerve biopsies.




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