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Biology

Cellesporing hjælp Photoconvertible proteiner under zebrafisk Development

doi: 10.3791/4350 Published: September 28, 2012

Materials

Name Company Catalog Number Comments
Materials:
Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).
Stereomicroscope Leica Lecia MZ6 Stereomicroscope Equipped with laser operating at 488 nm
Confocal laser scanning microscope Zeiss Zeiss LSM 710 NLO equipped with lasers operating at 488 nm, 561 nm, and 405 nm
Heating block
Pair of Dumont #5 forceps
Cover-glass-bottomed culture dish MatTek Corp Ashland MA USA
Microloader Pipette Tips Eppendorf
Vortex
needles
Reagents:
Egg water medium
60 μg/ml Instant Ocean Sea Salts in ddH2O
E3 medium
5 mM NaCl
0.17 mM KCl
0.33 mM CaCl2
0.33 mM MgSO4
0.00001% (w/v) Methylene Blue
4 mg/ml Tricaine stock solution
400 mg Tricaine (3-amino benzoic acidethylester) Sigma-Aldrich
97.9 ml ddH2O
2.1 ml 1M Tris (pH9)
Adjust at pH 7, aliquot and store -20 °C
1% LMA (Low melting temperature agarose, Lonza)
1g LMA/100 ml E3 medium 1X
Aliquot and store at 4 °C

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References

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  6. Hatta, K., Tsujii, H., Omura, T. Cell tracking using a photoconvertible fluorescent protein. Nat. Protoc. 1, 960-967 (2006).
  7. Stark, D. A., Kulesa, P. M. An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model. Dev. Dyn. 236, 1583-1594 (2007).
  8. Kulesa, P. M., Teddy, J. M., Stark, D. A., Smith, S. E., McLennan, R. Neural crest invasion is a spatially-ordered progression into the head with higher cell proliferation at the migratory front as revealed by the photoactivatable protein, KikGR. Dev. Biol. 316, 275-287 (2008).
  9. Nowotschin, S., Hadjantonakis, A. K. Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos. BMC Dev. Biol. 9, 49 (2009).
  10. Griswold, S. L., Sajja, K. C., Jang, C. W., Behringer, R. R. Generation and characterization of iUBC-KikGR photoconvertible transgenic mice for live time-lapse imaging during development. Genesis. 49, 591-598 (2011).
  11. Baker, S. M., Buckheit, R. W. 3rd, Falk, M. M. Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes. BMC Cell Biol. 11, 15 (2010).
  12. Watanabe, S., Punge, A., Hollopeter, G., Willig, K. I., Hobson, R. J., Davis, M. W., Hell, S. W., Jorgensen, E. M. Protein localization in electron micrographs using fluorescence nanoscopy. Nat. Methods. 8, 80-84 (2011).
  13. Habuchi, S., Tsutsui, H., Kochaniak, A. B., Miyawaki, A., van Oijen, A. M. mKikGR, a monomeric photoswitchable fluorescent protein. PLoS One. 3, e3944 (2008).
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Cellesporing hjælp Photoconvertible proteiner under zebrafisk Development
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Cite this Article

Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. Cell Tracking Using Photoconvertible Proteins During Zebrafish Development. J. Vis. Exp. (67), e4350, doi:10.3791/4350 (2012).More

Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. Cell Tracking Using Photoconvertible Proteins During Zebrafish Development. J. Vis. Exp. (67), e4350, doi:10.3791/4350 (2012).

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