Fluorescence In Situ Hybridization of Gut Bacteria in Mouse Intestinal Sections

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