Fluorescence In Situ Hybridization of Gut Bacteria in Mouse Intestinal Sections

Abstract

Source: Ng, K. M., Tropini, C. Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging. J. Vis. Exp. (2021).

This video demonstrates the procedure of fluorescence in situ hybridization (FISH) to stain gut microbiota within mouse intestinal tissue by hybridizing fluorescent probes to bacterial rRNA (ribosomal ribonucleic acid) and counterstaining the host structures for microscopic analysis.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee

1. Bacterial staining with FISH (Fluorescence in situ hybridization)
   1. Use a liquid blocker or PAP pen (hydrophobic barrier pen) to limit the area of liquid expansion by circling the area of each tissue section. Ensure that the ink does not come in contact with the section itself. Create a circle as close to each tissue section as possible without contacting the tissue to minimize the surface area that needs to be covered by the hybridization solution.
   2. Prepare the hybridization solution: for every 50 µL of pre-warmed hybridization solution, add 0.5 µg probe (e.g., 0.5 µL of 1 µg/µL probe). Pipette the hybridization solution onto the sections on the slide (~20 µL/section, depending on the section/circle size). Protect the hybridization solution and the slides from light from this point onward during incubation steps and storage.
   3. Ensure that the volume of liquid used covers the entire section upon overlaying with a flexible plastic coverslip. Incubate the slide in a humid chamber to reduce evaporation. Create a humid chamber with a pipette tip box with wipes or paper towels at the bottom that have been soaked with excess hybridization solution or phosphate-buffered saline (PBS) to provide humidity. Incubate the sections at 45-50 °C, depending on the probe set, for >3 h.
   4. Warm up the FISH washing buffer: 20 mM Tris-HCl, pH 7.4; 0.9 M NaCl to 50 °C during the incubation period. Prepare enough washing buffer to cover the slides in the Coplin jar.
   5. Remove the plastic coverslips; incubate the slides in FISH washing buffer in a Coplin jar, both pre-warmed to 50°C. Place the Coplin jar back into the 50°C oven for 10-20 min. For thick samples, remove the coverslips in the Coplin jar by adding the buffer and gently dislodging the coverslips to avoid smearing the slice.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Coplin jar	Fisher	08-813E
Cover glass, 22 × 22 mm, no #1	VWR	CA48366-227-1
DAPI	Sigma-Aldrich	D9542	Resuspended to 5 mg/mL
Empty pipette tip box(es)			To melt paraffin
FISH probes
FISH hybridization solution	--	--	20 mM Tris–HCl pH 7.4, 0.9 M NaCl, 0.01% (w/v) SDS in nuclease-free water. Optional addition of 5–50% (v/v) formamide
FISH washing buffer	--	--	20 mM Tris–HCl pH 7.4, 0.9 M NaCl
Fluorescently-labeled Ulex Europaeus Agglutinin (UEA-1) lectin	Vector Laboratories	FL-1061 (Fluorescein-labeled) or RL-1062-2 (Rhodamine-labeled)	UEA-1 labels fucosylated glycans, so it is not appropriate for germ-free mice or for samples from fucosyltransferase 2 (FUT2)-deficient hosts.
Forceps
HybriSlip flexible plastic cover slips, 22 x 22 mm × 0.25 mm	Sigma Aldrich	GBL722222	for FISH hybridization steps, can substitute glass coverslips
ImmEdge Hydrophobic Barrier PAP Pen	Vector Laboratories	H-4000
Kimwipes
Multipurpose Specimen Storage Containers, 473 mL	Fisher	14-955-117A	Can substitute with a glass or polyethylene container of your choice. Methacarn is not compatible with polystyrene or metal.
Nail Polish	Electron Microscopy Sciences	72180	Any nail polish should work
Oven			Necessary range: 45-60 °C
PBS, Phosphate-buffered Saline (10x solution)	Fisher	BP3991	Dilute to 1x for protocol
Sodium chloride	Fisher	S271
Trizma hydrochloride solution, 1M Tris-HCl, pH 7.4	Sigma-Aldrich	T2663-1L
Water, nuclease-free	Fisher	BP2484100