NE102 Lab Write-Up #2

B the nuclei of the untreated pc12 cells dyed with

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Egr1 in PC12 cells without treatment with NGF. b) The nuclei of the untreated PC12 cells dyed with DAPI. c) Egr1 in PC12 cells after one-hour treatment with NGF. d) The nuclei of the PC12 cells treated with NGF dyed with DAPI. Figure 2. Light micrographs of the localization of Neurofilament L (NFT L) in PC12 cells both treated and untreated with NGF. a) NFT L in PC12 cells without treatment of NGF. b) The DAPI- dyed nuclei of the PC12 cells without treatment of NGF. c) NFT L in PC12 cells after a two-day treatment of NGF. d) The DAPI-dyed nuclei of the PC12 cells with a two-day treatment of NGF.
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6 Figure 3 . Light micrographs of PC12 cells treated and untreated with NGF at objective of 20x and 40x. a) Undifferentiated PC12 cells at 20x. b) Undifferentiated PC12 cells before treatment with NGF at 40x. c) Differentiated PC12 cells after treatment with NGF at 20x. d) Differentiated PC12 cells at 40x. Results The original hypothesis behind the presence of NFT L and Egr1 after treatment with NGF was that the levels of these proteins would increase. Based on our data, our hypotheses were accurate. Figures 1 and 2 show the results from the experiment and Figure 3 distinctly illustrates the growth of PC12 cells after being treated with NGF. For the Egr1 protein, we did not get the correct results the first time the experiment was performed because the antibodies did not work as they were supposed to. In our incorrect data, the green fluorescent light in the images were appearing everywhere in the cell. We know this to be false because Egr1 is a transcription factor and should be found in the nucleus of the cell. This is a consequence of non-specific binding by the antibodies. This means that they bound to other molecules in the cell aside from the transcription factor, Egr1.
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7 Fortunately, Figure 1 displays results from ICC that did work properly. We were able to obtain and experiment on antibodies that binding properly to the protein. Image (a) depicts the cells without treatment of NGF and it is clear that Egr1 is not present. The dark circles present in the middle of the bright green fluorescent color are nuclei. When looking at image (b), we were able to conclude correctly that the dark circles in image (a) were then nuclei. When we switched the filter on the microscope, we were able to see images of the nuclei because they showed up as blue circles. This was due to the blue DAPI that the cells were dyed with. So wherever the dark circles were in image (a), we
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