ZFIN ID: ZDB-PUB-051025-1
High Throughput Expression Analysis of ZF-Models Consortium Clones
Thisse, C., and Thisse, B.
We perform a large scale analysis of gene expression by whole mount in situ hybridization during zebrafish embryogenesis and larva stages using templates provided by the ZFModels (zebrafish model for Human Development and Disease) consortium (European Community Integrated Project no. LSHG-CT-2003-503496).
Templates used are either cDNAs or specific exon sequences, PCR amplified from genomic DNA provided by Edwin Cuppen lab (Utrecht, Netherlands)
- Embryos from AB/TU fish (a strain generated from crosses of two wild-type lines, AB and TU) are collected, dechorionated by pronase treatment, allowed to develop at 28.5°C until the appropriate stage and then fixed by incubation over night in 4% paraformaldehyde at 4°C. Embryos older than 24h (hours post fertilization) are incubated in 0.3x Danieau medium supplemented with 1-phenyl-2-thiourea (PTU, 0.003%) to prevent accumulation of pigment. After fixation, embryos are dehydrated and stored at -20°C in 100% methanol prior to in situ hybridization.
The in situ hybridization is performed according to
Thisse, B et al (2004).
- The labeling reaction is monitored under a dissecting microscope and the reaction is stopped with 1x PBS at pH 5.5. Embryos are then mounted in 100% glycerol and incubated at least 24 hours in the dark at room temperature prior to observation. Embryos are mounted under a coverslip in 100% glycerol. Pictures are taken using a color CCD camera (Roper Scientific, Coolsnap) mounted on a dissecting microscope (Leica, M420) or on a compound microscope (Leica, DM RA2HC or Nikon, FXA).
Thisse, B., Heyer, V., Lux, A., Alunni, A., Degrave, A., Seiliez, I., Kirchner, J., Parkhill, J-P. and Thisse, C. (2004). Spatial and Temporal Expression of the Zebrafish Genome by Large-Scale In Situ Hybridization Screening. Meth. Cell. Biol. 77: 505-519
Acknowledging this work:
All clones, information, and images used from this work should cite this summary.
ERRATA and NOTES
In situ hybridizations for this high throughput analysis have been performed once. Mistakes may occur. Please contact C and B Thisse
if you detect anything wrong.