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Cellular resolution expression profiling using confocal detection of NBT/BCIP precipitate by reflection microscopy.

Gáspár Jékely1, Detlev Arendt

  • 1Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. jekely@embl.de

Biotechniques
|July 7, 2007
PubMed
Summary

This study introduces a new method to visualize gene expression in whole organisms using confocal reflection microscopy. This technique efficiently combines whole-mount in situ hybridization with high-resolution imaging for developmental biology research.

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Area of Science:

  • Developmental Biology
  • Microscopy Techniques
  • Molecular Biology

Background:

  • Determining 3D gene expression patterns at cellular resolution is crucial for developmental biology.
  • Traditional whole-mount in situ hybridization (WMISH) using nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) is sensitive but incompatible with high-resolution confocal laser-scanning microscopy (CLSM).

Purpose of the Study:

  • To develop a method for visualizing NBT/BCIP precipitate using CLSM for enhanced cellular resolution in gene expression studies.
  • To enable the combination of established WMISH techniques with advanced imaging for broader applications in model organisms.

Main Methods:

  • Utilized confocal reflection microscopy to visualize NBT/BCIP precipitate in WMISH samples.
  • Developed a simple WMISH protocol compatible with reflection CLSM.
  • Applied the method to model organisms including Drosophila, zebrafish, and Platynereis dumerilii.

Main Results:

  • Successfully visualized NBT/BCIP precipitate in WMISH samples using confocal reflection microscopy.
  • Demonstrated the compatibility of the protocol with fluorescent WMISH, immunostainings, and transgenic green fluorescent protein (GFP) marker lines.
  • Achieved double labeling of cell types and embryological structures.

Conclusions:

  • Whole-mount reflection CLSM provides efficient and high-resolution visualization of NBT/BCIP stained samples.
  • This technique facilitates large-scale cellular resolution expression profiling in both vertebrate and invertebrate model organisms.
  • The developed protocol enhances the integration of different imaging and labeling techniques for comprehensive developmental studies.