This study investigates the dynamics of prion-like proteins in transgenic nematodes using confocal microscopy. The proteins are tagged with a red fluorescent marker, allowing for real-time imaging of their transport within muscle cells.
Take transgenic nematodes that are maintained at the same developmental stage.
The body wall muscle cells of these nematodes express prion-like proteins, which are tagged with a red fluorescent protein and localized within acidic vesicles.
Prion-like proteins are misfolded, aggregating proteins that are associated with neurodegenerative diseases.
Next, transfer the nematodes onto an agarose pad containing polymeric nanoparticles and an anesthetic solution.
The combined effects of the nanoparticles and the anesthetic solution immobilize the nematodes on the agarose pad.
Place a coverslip over the agarose pad and seal it.
Now, position the agarose pad assembly with the nematodes under a confocal microscope and set the appropriate imaging parameters.
Acquire time-lapse images of the fluorescent-tagged proteins within the nematodes to monitor the vesicular transport of the prion-like proteins within and between the body wall muscle cells.
After preparing synchronized C. elegans control and prion domain transgenic lines, and 10% agarose pads according to the accompanying text protocol, pipette 3 microliters of 100 nanograms of polystyrene beads and 3 microliters of 4 millimolar levamisole into the agarose pad. Using a platinum wire pick, transfer worms at the desired age from a plate into the solution, and use a coverslip to cover.
To image the worms at the subcellular level, place the slide into the microscope slide holder of a confocal microscope, and use a 63X or 100X/ 1.4 NA oil objective. Next, open MetaMorph and select "Multidimensional Acquisition" and under the main tab, check the boxes for "Time lapse" and "Run Journals". Under the "Saving" tab, select or create the directory folder for saving files and assign a name to the file.
Under the "Wave Length" tab, use "YokoQuad Red" or an equivalent illumination of MRP imaging, and adjust the exposure to between 100 and 300 milliseconds, and the camera gain to between 100 and 300. Next, under the "Timelapse" tab, set the Time Interval to 1 second, "Duration" to 5 minutes, and the "Number of time points" to 301.
Under the "Journals" tab for Journal, select "AFC SET Z HOLD" and "AFC Return to Z HOLD". For Type, select "Special" twice, and for Initial Point, select "Start of time point" and "End of time point". When the setup is done, press "Acquire".
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