12/13/2023 0 Comments Axon to dendriteThen acquire a time lapse image set with acquisition occurring every 0.5 seconds for five minutes. Then to reduce photobleaching and phototoxicity, adjust the imaging parameters to maximize the signal to noise ratio and dynamic range using minimal exposure time and laser intensity. Now, using wide field epifluorescence find VAMP2 fluorent expressing cells through the oculars. Then switch from wide field illumination to TIRF illumination mode for imaging. Then replace the condenser and in the TIRF software sent the penetration depth to 110 nanometers. Place the condenser upside down on the optical bench so as not to scratch the lens.įine adjust the focal point of the laser on the ceiling and with the condenser removed, center the point to the center of the closed field diaphragm. ![]() Then in the imaging software, select the 491 laser illumination and open the shutter. ![]() Next, focus on the sample again in transmitted light illumination. Adjust the slider to 100 and then bring it back down to a value between 20 and 40. Imaging parameter optimization is important during imaging of floor intact exocytic vesicles to avoid focus drift and phototoxicity while producing images of high signal to noise ratio sufficient for analysis. Then set the refractive index of the sample and adjust the laser intensity by unchecking TTL for the 491 laser. Set the illumination to wide field and select the objective. Start the laser software and connect to the laser control software. After setting up the TIRF microscope and the sample and finding a neuronal focal plane according to the text protocol. Then carry out SDS page and Western blotting according to the text protocol. Invert the tubes intermittently to keep samples in solution. Then incubate one sample at 37 degrees Celsius for 30 minutes, and the other at 100 degrees Celsius for 30 minutes. Divide each experimental sample evenly into two tubes. Then, use homogenization buffer with 1%Triton X-100 and 5X sample buffer supplemented with BME to dilute the samples to a final concentration of 3-5 milligrams per milliliter. Next, use the Bradford assay to determine protein concentration. Transfer the lie state supernatant into a new chilled 1.6 milliliter tube on ice. Then centrifuge at 6, 010 RCF at 4 degrees Celsius for 10 minutes to pellet the non-solubilized material. Incubate the tubes on ice for two minutes to solubilize the proteins. Next, transfer the homogenized solution to a pre-cooled 1.6 milliliter microfuge tube on ice, and add 20%Triton X-100 to reach a final concentration of 1%Then with a 1, 000 microliter pipette, triturate the mixture ten times while minimizing bubbles. Incubate on ice for five minutes, and then using a cell lifter, homogenize the cells on ice.Īspirate the PBS from the next well of the same condition and add the recently homogenized mixture to the well which will increase protein concentrations. ![]() Aspirate PBS from the first well of a condition and replace with 250 microliters of homogenization buffer. The main advantage of this technique is that it combines live cell imaging to acquire both high spatial and temporal resolution in single cells and biochemical approaches to acquire population based information.Īfter culturing and simulating cortical neurons with Netrin-1 or a control according to the protocol text. This method can help answer key questions in the neuroscience field, such as when and where vesicle fusion inserts membrane material into the expanding plasma membrane during neuronal development. ![]() The overall goal of this procedure is to make quantifiable observations of the temporal and spatial occurrence of neuronal exocytic vesicle fushion and axon branching.
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