Immunofluorescence Lab

Post Lab Questions for Immunofluorescence

1.) In our experiment the primary antibody that was used was anti-tubulin, which was generated in the rabbit anti-mouse tubulin antibody. It is specific for the tubulin proteins to help identify their binding sites.

2.) The secondary antibody that was used was anti-actin, which was stained with FITC. It was generated in the goat anti-rabbit IgG. It is specific for the actin proteins. Adding a flurofore, which helps identify certain targets, better modified the secondary antibody.

3.) We used primary and secondary because they can be used simultaneously to identify different antibodies by switching light wavelengths. Whereas if only a primary fluorescent antibody were to be used some of the structures would not be seen. Only the first binding site of the antibodies would be visible, thus the other half of the bounded structure would be invisible.

4.)

The structure of the cell was very visible when using the anti-tubulin. When observing the anti-actin the microfilaments were visible. The actin filaments were much thinner than those of the microtubules. The actin filaments were best observed in the periphery of the cell compared to those of the tubulin, which are spread throughout the cell because they help stabilize the cell. The TRITC dyed cells depict better the whole structure of the cell, the nucleus is more visible than those that are dyed with FITC.

5.) On the controlled slide both microfilaments and microtubules are visible because it is treated with both anti-tubulin as well as anti-actin. The control is necessary because when observing just one of the antibodies you can go back and compare it and see if the structure correlate with what you