Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides.
We describe a detection principle for indirect fluorescence in situ hybridization (FISH) methods that with only one or two antibody layers dramatically improves FISH signal intensities. The method uses as a first layer an anti-hapten immunoglobulin [or (strept)avidin] conjugated to peroxidase. The quintessence of the method is the use of fluorochrome- or biotin labelled tyramides as peroxidase substrates to generate and deposit many fluorochrome or biotin molecules close to the in situ bound peroxidase. These may either be directly evaluated under the fluorescence microscope or after another incubation with fluorochrome-labelled (strept)avidin.
Raap
A K
AK
Department of Cytochemistry and Cytometry, Medical Faculty, Leiden University, The Netherlands.
van de Corput
M P
MP
Vervenne
R A
RA
van Gijlswijk
R P
RP
Tanke
H J
HJ
Wiegant
J
J
D000818:Animals; D001710:Biotin / Q000737:chemistry; D003374:Coumarins / Q000737:chemistry; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D010544:Peroxidases / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry; D012680:Sensitivity and Specificity; D014439:Tyramine / Q000031:analogs & derivatives* / Q000378:metabolism
D003374:Coumarins; D005452:Fluoresceins; D012235:Rhodamines; C005358:tetramethylrhodamine; D001710:Biotin; D010544:Peroxidases; D019793:Fluorescein; D014439:Tyramine