Dual-Fluorescence in situ Hybridization Protocol

Whole-mount in situ hybridization (WISH) was done according to the protocol published previously (12 (link)). Double-fluorescence in situ hybridization (dFISH) was performed adapting the WISH protocol using the TSA signal amplification system (PerkinElmer). In short, additional to the DIG-labeled ISH probe, a second fluorescein-labeled probe was generated using fluorescein labeling mix (Roche). Probe concentration was reduced to 0.02 ng/µL for both probes. Anti–fluorescein-HRP (PerkinElmer) and anti–DIG-HRP (PerkinElmer) were diluted 1:500. Signal amplification was done with amplification diluent fluorescein-tyramide and amplification diluent Cy3-tyramide (PerkinElmer) for 4 min each. Samples were mounted in Vectashield (Vector Laboratories) and pictures were taken with a Leica SP5 II confocal scanning microscope. Stacks were acquired sequentially and z-projected.

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Wunderer J., Lengerer B., Pjeta R., Bertemes P., Kremser L., Lindner H., Ederth T., Hess M.W., Stock D., Salvenmoser W, & Ladurner P. (2019). A mechanism for temporary bioadhesion. Proceedings of the National Academy of Sciences of the United States of America, 116(10), 4297-4306.

Publication 2019

fluorescein labeling mix Anti–fluorescein-HRP anti–DIG-HRP fluorescein-tyramide Cy3-tyramide Vectashield SP5 II confocal scanning microscope

Confocal scanning microscope Fluorescein Fluorescence in situ hybridization In situ hybridization Vector

Corresponding Organization : Universität Innsbruck

Other organizations : University of Mons, Innsbruck Medical University, Linköping University

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Variable analysis

independent variables

Probe concentration (reduced to 0.02 ng/µL for both probes)

dependent variables

Signal amplification with amplification diluent fluorescein-tyramide and amplification diluent Cy3-tyramide
Image acquisition with a Leica SP5 II confocal scanning microscope

control variables

WISH protocol published previously (12)
Anti–fluorescein-HRP and anti–DIG-HRP (both diluted 1:500)
Samples mounted in Vectashield (Vector Laboratories)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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