zebrafish staining

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Discussion and Conclusion

The staining of the motor neurons and the somites in the developing zebrafish is a difficult task. The zebrafish is a quickly developing organism, usually resembling the adult zebrafish after one day. The antibody for the motor neurons, will theoretically bind specifically to the developing motor neuron cells in the tail. However,given the small number of cells which are present at a given time, the resulting embryo does not show clear signs of the stained cells. In addition, a bigger factor may be the efficiency of the auto-fluorescent secondary antibody used. The cells of the zebrafish embryo often exhibit some auto fluorescence without any stimulation, so it may be difficult to differentiate the stained motor neurons from the surrounding cells, which also appear brightly stained.

The somites are also located in the tail region. They will eventually give rise to the vertebrae and the skeletal muscles in the back. The cell specific F6 antibody was used to stain the embryo for somite boundaries. However, little staining is observable because of the relatively similar appearance between the stained somite cells and surrounding cells, due to the secondary antibody resulting in stained cells which resembled surrounding cells.

The use of ZNP-1 and F6 coupled with the secondary antibody. Fluorescent Goat anti-mouse IgG+, to specifically stain the motor neurons and somites were not as successful as expected. The stain only resulted in few cells exhibiting a somewhat brighter color in the tail region,and was not conclusive. An alternate procedure using a horseradish peroxidase-conjugated secondary antibody, although more time consuming, resulted in a better signal-to-noise ratio and clearer staining.

References:

1. Gilbert, S.F. 2000. Developmental Biology, 6th ed. Sinauer Associates, Inc. Publishers. Sunderland, MA, p. 339-340, 451-452, 386, 389

2. Roy, M., V.E. Prince, and R.K. Ho, 1999) Heat shock produces periodic somitic disturbances in the zebrafish embryo. Mech. Dev. 85:27-34.

@ Cebra-Thomas, 2001

Last Modified: 2 August 2001

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		Discussion and Conclusion
		The staining of the motor neurons and the somites in the developing zebrafish is a difficult task. The zebrafish is a quickly developing organism, usually resembling the adult zebrafish after one day. The antibody for the motor neurons, will theoretically bind specifically to the developing motor neuron cells in the tail. However,given the small number of cells which are present at a given time, the resulting embryo does not show clear signs of the stained cells. In addition, a bigger factor may be the efficiency of the auto-fluorescent secondary antibody used. The cells of the zebrafish embryo often exhibit some auto fluorescence without any stimulation, so it may be difficult to differentiate the stained motor neurons from the surrounding cells, which also appear brightly stained. The somites are also located in the tail region. They will eventually give rise to the vertebrae and the skeletal muscles in the back. The cell specific F6 antibody was used to stain the embryo for somite boundaries. However, little staining is observable because of the relatively similar appearance between the stained somite cells and surrounding cells, due to the secondary antibody resulting in stained cells which resembled surrounding cells. The use of ZNP-1 and F6 coupled with the secondary antibody. Fluorescent Goat anti-mouse IgG+, to specifically stain the motor neurons and somites were not as successful as expected. The stain only resulted in few cells exhibiting a somewhat brighter color in the tail region,and was not conclusive. An alternate procedure using a horseradish peroxidase-conjugated secondary antibody, although more time consuming, resulted in a better signal-to-noise ratio and clearer staining. References: 1. Gilbert, S.F. 2000. Developmental Biology, 6th ed. Sinauer Associates, Inc. Publishers. Sunderland, MA, p. 339-340, 451-452, 386, 389 2. Roy, M., V.E. Prince, and R.K. Ho, 1999) Heat shock produces periodic somitic disturbances in the zebrafish embryo. Mech. Dev. 85:27-34.