The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.
The application of europium chelates as delayed fluorescent labels in FISH and immunocytochemistry is hampered by their relatively low quantum yield. To increase the intensity of the delayed fluorescence, we have used a recently introduced peroxidase-mediated amplification system. This system results in a large accumulation of biotin-tyramide, which is detected using streptavidin-europium chelate as label. Optimal staining conditions were evaluated for the immunocytochemical detection of vimentin in cryosections of rat liver, for DNA in situ hybridization (alphoid type probes and 40-KB cosmid probes), and for RNA in situ hybridization (detection of 28S ribosomal RNA, human elongation factor mRNA, and luciferase mRNA). Using a time-resolved fluorescence microscope, intense europium fluorescence was obtained in all these applications when the tyramide amplification system was applied. The signals were strong enough to be observed by eye using the microscope in the time-delayed mode. The routine application of this technique for localization and quantization of antigens or nucleic acid sequences in tissue exhibiting strong autofluorescence is discussed.
de Haas
R R
RR
Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.
Verwoerd
N P
NP
van der Corput
M P
MP
van Gijlswijk
R P
RP
Siitari
H
H
Tanke
H J
HJ
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis*; D001710:Biotin / Q000032:analysis*; D002614:Chelating Agents / Q000032:analysis*; D004247:DNA / Q000032:analysis; D005063:Europium / Q000032:analysis*; D005456:Fluorescent Dyes / Q000032:analysis*; D015200:Fluoroimmunoassay; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008099:Liver / Q000737:chemistry; D008156:Luciferases / Q000235:genetics; D008214:Lymphocytes / Q000737:chemistry; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D010445:Peptide Elongation Factors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D011817:Rabbits; D051381:Rats; D019809:Streptavidin; D016610:Tissue Embedding; D014439:Tyramine / Q000032:analysis*; D014746:Vimentin / Q000032:analysis
D001426:Bacterial Proteins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D014746:Vimentin; D005063:Europium; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D008156:Luciferases; D014439:Tyramine