Two will be stained with both primary and secondary antibodies to observe the condition of the microtubules and lysozomes, and the third sample will be treated with no primary antibodies, only secondary antibodies in order to ensure the secondary antibodies are not staining on their own. The remaining samples will be washed, by removing the nocodazole and adding fresh media. Two of these samples will be fixed after a time period of 15 minutes, two more will be fixed after 30 minutes, two more will be fixed after 45 minutes, and two more will be fixed after one hour. All the controls can be kept on one plate together, but the samples for each separate time period must be kept on different plates because they require incubation and must not be removed from the incubator until the proper amount of time has passed. To allow the cells to flatten out on the coverslips, they were left for three days before treatment with nocodazole.
Flat cells produce clearer images under the microscope.
The wash time periods were also altered, based on the observations from experiment 1. When viewed under the tubulin filter of the immunofluorescence microscope, the microtubules glow green because they are composed of this labeled tubulin. In each case, the condition of the microtubules can be observed as they are depolymerized by nocodazole then are washed and begin to regrow. The green strands show undisturbed microtubules. There are no longer long green strands where the microtubules used to be.