This article describes a protocol for using fluorescence in situ hybridization (FISH) to visualize bacterial 16S rRNA in mouse intestinal sections. The method involves hybridizing specific probes to the RNA, followed by counterstaining to highlight intestinal mucus and cell nuclei.
Begin by adding a solution containing bacterial 16S rRNA-specific Fluorescence in situ hybridization (FISH) probes to a slide containing the mouse intestinal sections colonized by bacteria.
Place a coverslip to spread the liquid containing probes and prevent evaporation.
Incubate the slide in a humidified chamber at an elevated temperature.
This unfolds the RNA secondary structure, facilitating hybridization of the probe with complementary sequences in bacterial 16S rRNA.
Remove the coverslip and incubate the slide in a pre-warmed washing buffer to remove unbound probes.
Rinse with a phosphate buffer to maintain physiological pH.
Apply a counterstain containing a mucus-specific dye and a nuclear dye, and incubate.
These dyes selectively bind to intestinal mucus and to the DNA of bacterial and host cells. Wash the slide with a buffer and mount coverslips onto the slide using mounting media.
Under a confocal microscope, observe distinct FISH-labeled bacteria, intestinal cells, and the mucus layer within the sections.
Create very close circles around each tissue section using a liquid blocker or PAP pen to limit the area of expansion needed to be covered by the hybridization solution, avoiding ink contact with the section.
Prepare the hybridization solution and add 0.5 micrograms of probe for every 50 microliters of the solution used. Pipette the solution onto the sections on the slide. Overlay the section with flexible plastic coverslips, ensuring that the volume of liquid used covers the entire section.
Create a humid chamber with a pipette tip box with wipes or paper towels that have been soaked with excess hybridization solution or PBS to provide humidity. Incubate the slide in the humid chamber at 45 to 50 degrees Celsius for at least three hours depending on the probe set to reduce evaporation. Remove the plastic coverslips, and incubate the slides in FISH washing buffer in a Coplin jar, both prewarmed to 50 degrees Celsius.
Place the Coplin jar back into the 50 degree Celsius oven for 10 to 20 minutes. Remove the FISH washing buffer and replace it with PBS in the Coplin jar. Immediately after refilling the Coplin jar with PBS, decant the PBS.
Remove the slides from the jar and pipette the counterstain on top of the entire section, while making sure to not touch the tissue with the pipette tip. Incubate at 4 degrees Celsius for 45 minutes. Wash the stains three times quickly with fresh PBS.
Wipe the back of the slides against a wipe or paper towel and let most of the PBS evaporate off the sections aided by a vacuum line connected to a pipette tip. Mount the sections using a mounting medium. Affix the coverslips to the slide by painting along the edges of the coverslip with clear nail polish, taking care to stay away from the edge of the slide. Let it set at room temperature.
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