I recently started working with zebrafish and confocal microscopy. My samples are double transgenic for gfap_mcherry and pcna_gfp. gfap labels Müller glia in the retina, and pcna is a proliferation marker facilitating DNA polymerase during proliferation. I should start imaging at d3 using multiple confocal microscopes to decide the most suitable one but I got very diffused gfp signal with all microscopes. What can be the reason for that?
If your PCNA-GFP signal is diffused in zebrafish larvae during confocal imaging, several factors could be contributing:
- ure and proper frame settings.
c. Zebrafish Autofluorescence
- Larvae (especially at older stages) can have high autofluorescence that masks GFP signal. Use spectral unmixing or careful filter selection.
d. Fixation (if fixed sample)
- GFP fluorescence is sensitive to fixation. Paraformaldehyde can reduce signal unless optimized. Live imaging is usually better for GFP.
🔬 3. Biological Considerations
a. Cell Proliferation Status
- PCNA is a proliferation marker; it’s only expressed during S-phase. In non-proliferative or quiescent tissues, you won’t see signal.
b. PCNA Localization
- PCNA typically shows nuclear punctate patterns during S-phase. Are you imaging at high enough resolution to detect nuclear foci?
c. Fusion Protein Stability
- GFP may destabilize PCNA or interfere with its localization. You can try adding a linker (e.g., GGSGG) between PCNA and GFP to improve function.
✅ Suggested Controls and Solutions
- Positive control: Express a plain GFP under the same promoter to confirm expression and imaging setup.
- Use EdU or BrdU labeling: To confirm whether cells are proliferating.
- Check with Western blot or qPCR: To confirm PCNA-GFP expression at the protein or transcript level.
- Imaging settings: Confirm with known GFP-expressing samples (e.g., Tg(actb2:GFP)).
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