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Your search keyword '"Söll, Iris"' showing total 10 results
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10 results on '"Söll, Iris"'

6. Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

Author

Hauptmann, Giselbert, Söll, Iris, Krautz, Robert, and Theopold, Ulrich

Subjects
INT, fluorescein, Fast Blue, fruit fly, Embryonic Development, Gene Expression, Gene Expression Regulation, Developmental, WISH, RNA Probes, Immunohistochemistry, biotin, azo dye, Issue 107, Animals, Fast Red, Drosophila, RNA, Messenger, alkaline phosphatase substrate, Digoxigenin, In Situ Hybridization, Developmental Biology
Abstract

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Published
2016

10. Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced Tyramide Signal Amplification and Its Combination with Immunofluorescent Protein Visualization in Zebrafish.

Author

Lauter G, Söll I, and Hauptmann G

Subjects
Alkaline Phosphatase metabolism, Animals, Embryo, Nonmammalian metabolism, Horseradish Peroxidase metabolism, Immunoassay, RNA, Messenger metabolism, Zebrafish embryology, In Situ Hybridization, Fluorescence methods, Zebrafish metabolism
Abstract

Fluorescent in situ hybridization (FISH) provides sensitive detection and visualization of RNA transcripts in tissues and cells with high resolution. We present here a multiplex RNA FISH method using enhanced tyramide signal amplification (TSA) for colocalization analysis of three different transcripts in intact zebrafish brains. To achieve enhancement of fluorescent signals, essential steps of the FISH procedure are optimized including embryo permeability, hybridization efficacy, and fluorogenic TSA-reaction conditions. Critical to this protocol, the enzymatic peroxidase (PO) reactivity is significantly improved by the application of viscosity-increasing polymers, PO accelerators, and highly effective bench-made tyramide substrates. These advancements lead to an optimized TSA-FISH protocol with dramatically increased signal intensity and signal-to-background ratio allowing for visualization of three mRNA transcript patterns simultaneously. The TSA-FISH procedure can be combined with immunofluorescence (IF) to compare mRNA transcript and protein expression patterns.

Published
2020
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