Abstract
BackgroundIn recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization. Despite the benefits of this technique, visualization of overlapping transcript distributions by differently colored precipitates remains difficult because of masking of lighter signals by darker color precipitates and lack of three-dimensional visualization properties. Fluorescent detection of transcript distributions may be able to solve these issues. However, despite the use of signal amplification systems for increasing sensitivity, fluorescent detection in whole-mounts suffers from rapid quenching of peroxidase (POD) activity compared to alkaline phosphatase chromogenic reactions. Thus, less strongly expressed genes cannot be efficiently detected.ResultsWe developed an optimized procedure for fluorescent detection of transcript distribution in whole-mount zebrafish embryos using tyramide signal amplification (TSA). Conditions for hybridization and POD-TSA reaction were optimized by the application of the viscosity-increasing polymer dextran sulfate and the use of the substituted phenol compounds 4-iodophenol and vanillin as enhancers of POD activity. In combination with highly effective bench-made tyramide substrates, these improvements resulted in dramatically increased signal-to-noise ratios. The strongly enhanced signal intensities permitted fluorescent visualization of less abundant transcripts of tissue-specific regulatory genes. When performing multicolor fluorescent in situ hybridization (FISH) experiments, the highly sensitive POD reaction conditions required effective POD inactivation after each detection cycle by glycine-hydrochloric acid treatment. This optimized FISH procedure permitted the simultaneous fluorescent visualization of up to three unique transcripts in different colors in whole-mount zebrafish embryos.ConclusionsDevelopment of a multicolor FISH procedure allowed the comparison of transcript gene expression domains in the embryonic zebrafish brain to a cellular level. Likewise, this method should be applicable for mRNA colocalization studies in any other tissues or organs. The key optimization steps of this method for use in zebrafish can easily be implemented in whole-mount FISH protocols of other organisms. Moreover, our improved reaction conditions may be beneficial in any application that relies on a TSA/POD-mediated detection system, such as immunocytochemical or immunohistochemical methods.
Highlights
In recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization
Effects of viscosity-increasing polymers and substituted phenol compounds Despite using the tyramide signal amplification (TSA) system for signal enhancement in fluorescent in situ hybridization (FISH) applications, the results can be dissatisfying with respect to signal sensitivity and signal-to-noise ratio
We found that addition of 5% dextran sulfate during hybridization led to a significant increase in signal intensity (Figure 1A, C)
Summary
In recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization. In the past decades, the examination of forebrain-specific regulatory gene expression patterns supported the development of a prosomeric concept of. Overlapping or colocalized expression is often difficult to resolve by chromogenic two-color in situ hybridization because of lower second round detection sensitivity, masking of the lighter red signal by the darker blue color precipitate, and lack of three-dimensional visualization possibilities. These limitations may be overcome by fluorescent in situ hybridization (FISH), which offers selective detection of different transcripts at high spatial resolution. Despite the increased sensitivity through tyramide signal amplification, POD substrate turnover is still limited by the relatively short reaction time compared to alkaline phosphatase, so that less abundant mRNA species may be still difficult to detect
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