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April 2012 Molecular Cell lab Embryonic Development

Introduction: One of advantages that the Zebrafish embryo is its translucency; it enables the investigator to examine its development readily with the dissecting microscope; there are different types of stages in the embryonic life of a zebrafish. There are the segmentation period, the pharyngula period, and the hatching period. We also learned four different stages through which the cell fate of pluripotent cells is progressively restricted during development: 1. Competence Cell can become a particular type of cell if exposed to the right environment 2. Specification Cell has received appropriate signal to become specific type of cell but fate can still be changed by other signals 3. Commitment (determination) Cell has entered differentiation pathway. It can become specific type of cell even in the present of inhibitory signals 4. Differentiation Cell expresses genes characteristic of a specific type of cell, ex. Neuron To visualize the embryo, we used immune-staining method. For their control, they did not add a secondary antibody. I hypothesized if the primary antibody which is the tubulin enters the embryo, it will illustrate the structure of the cytoskeleton.

Procedure: After removing the embryo from its shell we place them in a centrifuge tube that hold PBS and 2% Triton X. One week later, we than did the following: 1. Block with primary anti-body: Mouse tubulin 1/100 in PBS for 1 hour 2. Wash 3X 2 minute in PBS + 1% BSA + Triton X 3. Add secondary anti-body; goat anti-mouse- Alex 488; 45 minute in room temp

4. Wash 3X 2 minute in PBS + 1%BSA + Triton 5. 100% glycerol 5 Min; 100 micro- Liter 6. Add Dapi (Half a drop) to glycerol: wait 5 minutes 7. Mount Embryos

Results:

Control Antibody

Antibody- DIC

Antibody Fluorescent

Discussion: The primary figure shows the control antibody, they only used a secondary antibody. Te embryo observed show a more complete structure DIC (differential Interference Contrast Microscopy) image (fig. 2) showed the morphology of the embryo. Some mistakes was observed as they were seeing them under the microscope, maybe the staining was not as successful. The blue DAPI (used to stain the nucleus) stain showed nothing as was expected. To conclude, if the experiment didnt include experimental error, it would have been successful. We obtained a fully form embryo with the tubulin showing the complete cytoskeleton, we did observe indications of its formation. Hence, to complete this experiment for better result, we need a little more practice.