This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.
This method can help answer key questions in the C.elegans field, such as cell biology. The main advantage of this technique is that it is fast and efficient to obtain signals of nuclear staining, GFP, and sub-cellular localization changes simultaneously. To begin, generate an extrachromosomal array align with a translational construct of target genes.
Alternatively, obtain such lines from the Caenorhabditis Genetic Center, which is a public resource for C.elegans research. Or, from a previously a previously published research laboratory. After exposing worms to gamma radiation to integrate the extrachromosomal lines into the genome, select the stably expressed lines so that each animal expresses a marker protein such as a GFP or roller.
To remove mutations generated during the radiation, outcross the lines at least two times. Use the resulting transgenic line to detect protein expression pattern changes under stress. Next, after preparing NGM worm plates according to the text protocol, streak an OP50 clone and culture the bacteria in liquid LB broth overnight.
Seed the NGM plates with liquid OP50 culture and incubate the plates in a 37 degree Celsius incubator overnight to grow bacteria lawn as a food source for the worms. Cool down the plates at room temperature for at least 30 minutes before use. Grow the transgenic line at 20 degrees Celsius without starvation, for at least two generations prior to the heat stress assay.
Pick eight to ten young, gravid adults to a new worm plate. Then let them lay eggs for about three to five hours, and remove all the adults from the plates. To synchronize the worms, let the eggs hatch and grow the larvae for approximately 48 hours to the fourth larval, or L4 stage.
That can be identified by the vulva, which is a half-moon structure. Then heat-stress the worms by incubating the plates with the L4 larvae at 35 degrees Celsius for two to five hours. Include a thermometer in an incubator to accurately measure the temperature.
Pipette 10 microliters of M9 onto the center of a glass slide. Then pick heat-shocked worms and transfer them into the drop of buffer. Alternatively, use M9 to wash the worms off the plate.
Then centrifuge the sample at 1000 times gravity at room temperature for one minute. Discard the supernatant, then add the worms to the glass slide. Under a dissecting microscope, use a soft tissue to drain any excess liquid.
Then, while still under the microscope, add 10 microliters of 95%alcohol onto the worms and continue to observe them. Immediately after the ethanol dries, repeat the addition of ethanol two more times. It is critical towards the process of the ethanol evaporating from the animals under the dissecting microscope.
Once the ethanol comes off the animals, immediately proceed to the next step. Add 10 microliters of DAPI to the worms. Immediately apply a cover slip and seal the slide with transparent nail polish gel.
After allowing the nail polish to set for approximately 10 minutes, view the animals under a fluorescence microscope. To fix animals with acetone, use M9 to wash off the heat-shocked plate and centrifuge the sample at 1000 times gravity at room temperature for one minute. Discard the supernatant and use one milliliter of distilled water to wash the pellet.
After removing the supernatant, add 400 microliters of 30%acetone and incubate the sample for 15 minutes. Then centrifuge the tube at 1000 times gravity at room temperature for one minute. After discarding the supernatant, use 500 microliters of distilled water to wash the animals two times.
Add 200 microliters of DAPI to the pellet, or four times the amount of the total worms'volume to the tube, and incubate the sample for 15 minutes. Centrifuge the sample and discard the supernatant. Then use 500 microliters of distilled water to wash the pellet two times before mounting and imaging as before.
To carry out imaging, turn on the UV light source and the fluorescence microscope. Then turn on the computer connected to the microscope. Load the slide onto the fluorescence microscope.
Use a 10X objective lens to locate the worms. Then increase the magnification power to 400X and focus on the specimen. Next, open the software provided with the microscope.
Click on Acquisition, then click on Camera, and select the camera connected to the microscope. Under the same Acquisition, click on Multidimensional Acquisition. This opens a new Multidimensional Acquisition navigation window.
Click the multi channel button and all available channels will appear. Choose blue, green, and DIC to observe the specimen. In the Multidimensional Acquisition navigation window, leave the blue DAPI, green GFP, and gray DIC channels on, and right-click on other channels, such as red Rhodamine and white Brightfield, to close them.
To measure the exposure time for each channel, left-click the blue DAPI channel to select it and click measure"to take a live image with an automated exposure time. On the left side of the live image window, manually adjust the exposure time if desired, then click OK.Set up the exposure times for the GFP and DIC channels in a similar manner. In the Multidimensional Acquisition navigation window, click on Start"to automatically collect three images under the three different channels, in order, with the set exposure times.
To save the file, in the main menu, click on File, Save As.Input the file name and the folder. Save the file as the default file format to preserve the detailed imaging information for future reference. To export the file in other formats, under File, click Export.
Set the file name and folder. Check Create a project folder"to better organize the data. After choosing a desired file type from the drop-down menu, click Start"to export.
There are two chloride intracellular channel protein homologues in C.elegans, exl-1 and exc-4. A recent study showed that exl-1 regulates animal stress management. Here, a translational exl-1 GFP construct was integrated into an animal genome, and transgenic lines were created, which stably express green fluorescent exl-1.
Under heat stress, exl-1 GFP accumulates in the nucleus in the intestinal region as a strong GFP signal overlaps with the nucleus. To confirm the subcellular localization of exl-1, DAPI staining was carried out in both ethanol and acetone fixed animals. Both methods show clear nuclei in the animal body and the overlapping GFP and DAPI signals confirm that exl-1 GFP indeed accumulated in the nuclei in the intestinal region.
Once mastered, this technique can be done in one hour if it is performed properly. After watching this video, you should have a good understanding of how to perform a quick fixation to preserve the GFP signal and protein nuclear translocation under stress in C.elegans.
