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| Content Provider | Springer Nature : BioMed Central |
|---|---|
| Author | André, Maëlys Dinvaut, Sarah Castellani, Valérie Falk, Julien |
| Abstract | Background Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. Results We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. Conclusions We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology. |
| Related Links | https://bmcbiol.biomedcentral.com/counter/pdf/10.1186/s12915-024-01922-0.pdf |
| Ending Page | 16 |
| Page Count | 16 |
| Starting Page | 1 |
| File Format | HTM / HTML |
| ISSN | 17417007 |
| DOI | 10.1186/s12915-024-01922-0 |
| Journal | BMC Biology |
| Issue Number | 1 |
| Volume Number | 22 |
| Language | English |
| Publisher | BioMed Central |
| Publisher Date | 2024-06-03 |
| Access Restriction | Open |
| Subject Keyword | Life Sciences RNA fluorescence in situ hybridization (HCR RNA-FISH) Immunofluorescence (IF) Tissue clearing ECi Whole mount chicken embryos Development Gene expression Lightsheet microscopy |
| Content Type | Text |
| Resource Type | Article |
| Subject | Ecology, Evolution, Behavior and Systematics Structural Biology Biochemistry, Genetics and Molecular Biology Plant Science Biotechnology Physiology Agricultural and Biological Sciences Cell Biology Developmental Biology |
| Journal Impact Factor | 4.4/2023 |
| 5-Year Journal Impact Factor | 5.4/2023 |
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