Lasers have made the 21st century even more technologically advanced with the implementation at the cellular level. Confocal microscopy alternatively to ultraviolent mercury lamps, are higher in wavelength and are more detailed in illumination in regards to shapes of organelles (Katula). In this experiment, using confocal microscopy, two organelles were visualized to understand the drug effect of nocodazole on NIH3T3 cells. Using two fluorescent stains, Mitotracker Red (accumulates in mitochondria) and Hoechst 33342 (accumulates in nuclei) were used to test what microtubules would look like resulting from the drug. Nocodazole is a drug that penetrates the microtubules to where it can be synchronized. (Karbowski). Microtubules are made up of polarized tubulin that are important for cytoskeletal rigidity, mitotic division, and act as a branch in transporting molecules to other parts of the cell. In the following experiment, nuclei and mitochondria were focused under a confocal microscope to visualize what cell structure and shape would be obtained before and after treatment of drug. Polarized tubulin attach to nocodazole to measure effect of microtubule structure to see whether or not cell shapes become smaller and more round in shape as opposed to controlled microtubule. Damaged Microtubules will make cell shapes round and Mitochondria will become clumped at only one p art of NIH 3T3 cell.
Methods.
In comparing controlled NIH3T3 cells with treated NIH3T3 cells, both cells were stained with Mitotracker Red and incubated under to ensure enough time for stain penetration to cell. After red medium was extracted from cell, paraformaldehyde fixative was added and removed multiple times to ensure cells were killed off but still maintained cellular structure and conformational shape. Cells were incubated once more before an additional application of fixative. Cells were then stained with Hoeschst 33342 and fixative before being placed on shaker for sustainable amount of time.