The fluorescence of control LLC-Pk1 cells was distributed indiscriminately throughout the cellular components due to the absence of primary antibody for the fluorescently tagged secondary antibody to bind to. The lack of affinity for a specific target within or around the cell allowed for the fluorescently tagged secondary antibody to freely bind to any structure or surface that it desired. This resulted in a fluorescent image in which most cellular structures and area surrounding the cells was tagged with fluorescent dye, ultimately brightening much of the area under the cover slip and outputting a low fluorescence intensity in the area of interest (tubulin). The fluorescence of the indirect immunofluorescence stained cells localized specifically to tubulin structures due to the presence of a primary antibody which targeted antigens located on tubulin molecules within the cells. This allowed for multiple secondary antibodies (which possess a high binding affinity for the primary antibodies) to bind to the primary antibodies, achieving a highly amplified fluorescence intensity on only tubulin structures. This is visually represented in Figures 1 and 2, and quantitatively measured in Table 1. Direct targeting of the actin cytoskeleton in LLC-Pk1 cells and NIH 3T3 cells allowed for clear visual distinctions to be seen in the structural composition of the actin cytoskeleton in each cell type. The actin cytoskeleton of the NIH 3T3 cell group is considerably more spread out within the cell, rather than localized to one region of the cell. This is due to the large role that fibroblasts play in maintaining structural integrity of connective tissues in living organisms. These cells must possess qualities of strength and durability throughout the cell, which is provided by the widely distributed actin cytoskeleton. Additionally, fibroblasts are able to migrate as individual cells, so the actin cytoskeleton extending within the branched structure could aid mobility on the cellular level. This structure can be visualized in Figures 3 and 4. Conversely, the actin cytoskeleton of the epithelial cells is more localized around the nucleus of the cell, rather than spread throughout the cell. This can be explained by the epithelial cells inability to migrate as individual cells, so a widely distributed actin cytoskeleton is not necessary. Additionally, the localization of the actin cytoskeleton specifically around the nucleus results from the large role that the actin cytoskeleton plays in cell division by transporting cellular components and preparing the cell to divide. This localization can be seen in Figures 3 and 4.
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