RNA FISH staining is useful for the visualization, identification, and quantification of microbes that naturally colonize or infect C.elegans, including microbiome bacteria and intracellular pathogens. This technique is simple, quick, and robust, allowing one to effectively stain and visualize microbes in the context of a whole intact animal. We can use RNA FISH to detect and visualize gut microbiome bacteria in wild C.elegans.
This can help us understand comparable interactions in mammalian systems. After transferring nematodes with the desired microbe of interest, from NGM plates to microcentrifuge tubes, wash the nematodes by adding one milliliter of 1X PBST to microfuge tubes. Spin down the samples in a microcentrifuge or Nanofuge at the appropriate speed.
Using a pipette, remove all but 100 microliters of the supernatant. Repeat washing with PBST two to three times. To fix the nematodes with paraformaldehyde, start by adding 33 microliters of 16%paraformaldehyde to the microfuge tube containing 100 microliters of supernatant above the nematode pellet for a final concentration of 4%paraformaldehyde.
Incubate the samples containing the nematodes colonized or infected with the microbe of interest for 30 to 45 minutes at room temperature. Remove the paraformaldehyde solution and add 0.5 milliliters of PBST to the samples. Spin down the samples in a microcentrifuge at the appropriate speed as mentioned in the text manuscript.
With a pipette, remove as much of the supernatant as possible without disturbing the pellet. Add 0.5 milliliter of PBST to the samples in the microfuge tubes. Repeat washing with PBST two to three times.
Following the last wash, spin down the samples and remove the supernatant, leaving the pellet undisturbed. Next, prepare one milliliter of hybridization buffer per sample. Add 800 microliters of hybridization buffer to the microfuge tubes containing the nematode pellet.
Pellet the samples in the microcentrifuge. Remove the supernatant without disturbing the pellet. Prepare 100 microliters per sample of hybridization buffer with the desired FISH probe to a final concentration of 5 to 10 nanograms per microliters probe and vortex to mix.
Add 100 microliters of hybridization buffer containing FISH probe to each sample. Mix by gently flicking or inverting the tubes. Incubate the samples overnight in a dry bath at 46 to 54 degrees Celsius or a thermal mixer at 46 to 54 degrees Celsius at 1, 200 rotations per minute.
Prepare three milliliters of wash buffer per sample. Centrifuge the samples at the appropriate speed. Remove the hybridization buffer using a pipette while being careful to leave the nematode pellet undisturbed.
Add one milliliter of prepared wash buffer to each sample. Centrifuge the samples at the appropriate speed. Remove the wash buffer using a pipette, while being careful to leave the pellet undisturbed.
Add one milliliter of prepared wash buffer to each sample. Incubate the samples for one hour at 48 to 56 degrees Celsius in a dry bath or thermal mixer at 48 to 56 degrees Celsius at 1, 200 rotations per minute. If incubating in a dry bath, gently invert the tubes every 15 to 20 minutes.
Centrifuge the samples at the appropriate speed. Using a pipette, remove the wash buffer, while being careful to leave the pellet undisturbed. Add 100 to 500 microliters of PBST to each of the samples.
At this point, the samples can be stored in PBST at four degrees Celsius for up to a week until the protocol is ready to be continued. Pellet the samples at the appropriate speed. Remove as much PBST as possible without disturbing the nematode pellet.
Add 20 microliters of anti-fade mounting medium with DAPI to the samples. Load a 20 microliters pipetter with a 200 microliters pipette tip and use scissors to cut the tip of the pipette off to allow larger nematodes to be pipetted. With the cut pipette tip, transfer 5 to 10 microliters of the pellet onto a microscope slide.
Cover with a 22 by 22 coverslip. To store the slides, seal the edges of the coverslip with nail polish and keep them in a dark box at four degrees Celsius until ready for further use. Under the differential interference contrast microscope, a wild Caenorhabditis tropicalis strain was found to be colonized with a bacterium that appears to directionally adhere to the intestinal epithelium.
Fluorescent signal from the green universal 16S rRNA probe and the red Alphaproteobacteria species probe overlap completely, suggesting that most bacteria colonizing the intestines are the adhering Alphaproteobacteria bacterium. The bipartite RNA genome of the Orsay virus consists of RNA1 and RNA2 segments and FISH probes targeting both segments have been developed. ZIP1 protein tagged with green fluorescent protein is seen localized in the intestinal nuclei of cells that show positive Orsay virus FISH staining in the cytoplasm.
Multiple animals stained with Orsay-specific or N.parisii-specific FISH are shown to indicate the strength of this signal for easy quantification. Co-infection of C.elegans with two microsporidia parasites using species-specific probes that compete for binding to the 18S, the use of DAPI to stain nuclei allows for better localization of the infection in the context of the whole animal, especially for the intestine which has large, easily identifiable nuclei. Infections with N.parisii result in the development of meronts into spores.
The resulting FISH staining demonstrates small and large rod-shaped structures that likely correspond with N.parisii spores, which are stained with N.parisii-specific probes in red. When selecting the fixative agent, remember that PFA fixation allows for better preservation of morphology and transgenic GFP, but acetone fixation is necessary for the visualization of sporoplasms. RNA FISH can be used to identify and visualize bacteria colonizing the C.elegans intestines.
Researchers can then perform more genetic screens to identify factors involved in these host-microbe interactions.
