Colorimetric in situ hybridization (CISH) with digoxigenin-labeled oligonucleotide probes in autofluorescent hyphomycetes.
We used digoxigenin-labeled probes for in situ hybridization of hyphomycetes to replace the commonly used fluorescent proof of probe binding by a colorimetric reaction. The resulting blue-purple, intracellular precipitate could be easily detected by light microscopy, and thus presented a promising method to overcome autofluorescence of fungal material and substratum. Optimal cell fixation and permeabilization procedures, as well as hybridization conditions were developed on the example of two different hyphomycetes: Phialophora sp. and Cartapip, a colorless mutant of Ophiostoma piliferum (Agra Sol).
Schröder
S
S
Department of Geomicrobiology, Institute of Chemistry and Biology of the Marine Environment, Carl von Ossietzky-University, Oldenburg, Germany. susanne.schroeder@mail.uni-oldenburg.de
Hain
M
M
Sterflinger
K
K
D011232:Chemical Precipitation; D003124:Colorimetry / Q000379:methods*; D004271:DNA, Fungal / Q000032:analysis*; D004076:Digoxigenin; D005453:Fluorescence; D017403:In Situ Hybridization / Q000379:methods*; D003904:Mitosporic Fungi / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes; D010676:Phialophora / Q000235:genetics / Q000302:isolation & purification; D016707:Tissue Fixation
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D004076:Digoxigenin