Home›Cell Biology›Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence
Cell BiologyJoVE (Open Access)Citable · DOI
Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence
DOI: 10.3791/56261-v
What you'll learn
✓Perform whole mount fluorescent in situ hybridization on zebrafish embryos
✓Apply combined FISH and immunofluorescence to visualize multiciliated cells
✓Mount and image zebrafish pronephros tissue for cilia analysis
Protocol
Biopharma Insights Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.
Difficulty
advanced
Total time
~3–4 days (including overnight hybridization and incubations)
Model organism
Danio rerio (zebrafish)
Biosafety
BSL-1
Steps
1
Fix embryos and perform in situ hybridization
Prepare zebrafish embryos through fixation and optimize conditions for probe hybridization to target ciliated cell markers.
▶ 00:50
2
Conduct hot washes and antibody blocking
Remove unbound probe, block nonspecific antibody binding sites, and incubate primary antibodies in maleic acid buffer.
▶ 03:05
3
Detect probe signal and perform immunofluorescence
Develop probe detection signal using peroxidase activity, quench enzyme activity, and apply fluorescent secondary antibodies.
▶ 04:27
4
Mount pronephros tissue and image multiciliated cells
Dissect zebrafish pronephros, mount tissue on slides, and acquire fluorescence microscopy images of labeled ciliated cells.