Co-localization of multiple antigens and specific DNA - A novel method using methyl methacrylate-embedded semithin serial sections and catalyzed reporter deposition

Citation
M. Mueller et al., Co-localization of multiple antigens and specific DNA - A novel method using methyl methacrylate-embedded semithin serial sections and catalyzed reporter deposition, AM J PATH, 157(6), 2000, pp. 1829-1838
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Research/Laboratory Medicine & Medical Tecnology","Medical Research Diagnosis & Treatment
Journal title
AMERICAN JOURNAL OF PATHOLOGY
ISSN journal
0002-9440 → ACNP
Volume
157
Issue
6
Year of publication
2000
Pages
1829 - 1838
Database
ISI
SICI code
0002-9440(200012)157:6<1829:COMAAS>2.0.ZU;2-X
Abstract
Co-localization of proteins and nucleic acid sequences by in situ hybridiza tion and immunohistochemistry is frequently difficult as the process necess ary to detect the target structure of one technique may negatively affect t he target of the other. Morphological impairment may also limit the applica tion of the two techniques on sensitive tissue. To overcome these problems we developed a method to perform is situ hybridization and immunohistochemi stry on semithin sections of methyl methacrylate-embedded tissue. Microwave -stimulated antigen retrieval, signal amplification by catalyzed reporter d eposition, and fluorescent dyes were used for both techniques, yielding hig h sensitivity and excellent: morphological preservation compared to convent ional paraffin sections. Co-localization of in situ hybridization and. immu nohistochemistry signals with high morphological resolution was achieved on single sections as well as on adjacent multiple serial sections, using com puterized image processing. The latter allowed for the co-localization of m ultiple antigens and a specific DNA sequence at the same tissue level. The method was successfully applied to radiation bone marrow chimeric rats crea ted by transplanting wild-type Lewis rat bone marrow into TK-tsa transgenic Lewis rats, in an attempt to trace and characterize TK-tsa transgenic cell s. It also proved useful in the co-localization of multiple antigens in per ipheral nerve biopsies.