NE102 Lab Write-Up #2

Incubation of formaldehyde we pipetted 1 ml of pbs

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incubation of formaldehyde, we pipetted 1 mL of PBS into each chamber and incubated for five minutes again. After that, we removed the PBS from each chamber and put it into the formaldehyde waste tube. We repeated the PBS wash two more times and discarded the PBS into the general waste beaker. We then incubated the cells in each chamber with 1 mL of 0.5% Triton X-100 for ten minutes. Again, the cells were washed with PBS three times. After the third wash, 500 µL of 5% NGS was put into each chamber using a P1000 pipetman. We incubated these cells for one hour. During the hour incubation period, we diluted the primary antibodies. In a 15 mL conical tube, we pipetted 2.1 mL of 5% NGS and 42 µL of Rb α-Egr1. We put this solution on a vortex and then kept them on ice until later use. After the hour incubation, we transferred 1 mL of Rb α-Egr1 into each chamber. These slides were kept at 4˚C. Addition of Secondary Antibody Before adding in the second antibody, we washed the cells in each chamber three times with PBS as directed above with five minute incubation periods between each wash. During this time, we diluted the secondary antibody, Goat α-Rabbit-AlexFluor488 in 5% NGS. To do this, 2.2 mL of 5% NGS was put into a 15 mL conical tube and then a
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4 P200 was used to transfer 22µL of Gt α-Rb-Alexa488 antibody into the tube. Following the third and final PBS wash, we used a P1000 to transfer 500 µL of the secondary antibody solution prepared in the previous step onto the PC12 cells. Another hour incubation period followed. Then we washed the cells with PBS three more times. Preparation of Glass Slides After the last PBS wash, we removed the plastic chambers from the glass slides using the removal device. We immediately added several drops of VectaShield to mount the medium onto each chamber of the glass slide. Then a glass cover slip was added on of the chambers and left for 15 minutes so the VectaShield dried. Fluorescence Microscopy We used a fluorescent microscope to obtain images of the cells at the 40x objective. We captured images of the NFT L and Egr1 cells that were both untreated and treated with NGF. We also took images of the treated and untreated cells that were dyed with DAPI to visualize the localization of the proteins in the cells.
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5 Figures Figure 1. Light micrographs of the localization of Egr1 in PC12 cells with and without treatment of NGF. a) Egr1 in PC12 cells without treatment with NGF.
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