RNA FISH in embryos was performed using fluorescence-labeled probes or digoxigenin-labeled probes detected by an anti-dig antibody and a fluorescence-labeled secondary antibody 58 (link). In our current study, probes were prepared from cDNA plasmids or genomic PCR products; for direct fluorescence labeling, the FISH Tag™ RNA kit (Alexa Fluor 488, Life Technology) was used. Embryos used in bcd and nos mRNA FISH were from 0–1-hr collections and those used in hb and eve mRNA FISH from 0–3-hr and 0–4-hr collections, respectively. Imaging was performed under the Zeiss Imager Z1 ApoTome microscope with a Zeiss Plan 10× Aprochromat objective. Imaging acquisition was performed under linear settings and data analysis (MATLAB, Math Works) was based on fluorescent intensities extracted from the cytoplasmic layer of midsagittal images as a function of AP position (for hb and eve) 58 (link), or as epifluorescence intensities (for bcd and nos) 23 (link).
Protocol detail
RNA FISH Imaging in Drosophila Embryos
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Alexa fluor 488
Anti antibody
Cdna
Cytoplasmic
Digoxigenin
Embryos
Fish
Fluorescence
Fluorescence antibody
Fluorescence probes
Genomic
Microscope
Mrna
Plasmids
Corresponding Organization :
Other organizations : Cincinnati Children's Hospital Medical Center, Chinese Academy of Sciences, Institute of Biophysics
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Variable analysis
independent variables
- Probe type (fluorescence-labeled probes, digoxigenin-labeled probes)
- Fluorescence intensity of mRNA signals for bcd, nos, hb, and eve
- Spatial distribution of mRNA signals along the anterior-posterior (AP) axis
- Developmental stage of embryos (0-1 hr, 0-3 hr, 0-4 hr collections)
- Imaging settings (linear settings, Zeiss Imager Z1 ApoTome microscope with Zeiss Plan 10× Aprochromat objective)
- Data analysis method (fluorescent intensities extracted from cytoplasmic layer of midsagittal images, epifluorescence intensities)
- Positive control: Not specified
- Negative control: Not specified
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