In Situ Detection of Bacteria in Mouse Gingival Tissue Sections

Take a mouse gingival section infected with pathogenic bacteria, pretreated to access bacterial DNA.

Apply a buffer with a digoxigenin-labeled DNA probe targeting bacterial DNA.

Place a coverslip and seal to prevent sample dehydration.

Introduce heat to denature the DNA, then incubate under hot and humid conditions to allow probe hybridization.

Chill the slide and remove the coverslip, and wash multiple times with buffer to remove unbound probes.

Add a blocking solution to prevent non-specific antibody binding.

Incubate with enzyme-conjugated antibodies that bind to the digoxigenin.

Wash to remove unbound antibodies.

Add an inhibitor to selectively inactivate the endogenous enzyme.

Apply a substrate that reacts with the enzyme to give a colored precipitate.

Rinse with water to remove excess substrate.

Add a dye to stain the nuclei.

Wash to remove excess dye.

Dehydrate in ethanol and treat with xylene to increase tissue transparency.

Mount the section.

Under a microscope, visualize the bacteria within the section.

Start the in situ by first immersing the slides in 2x SSC for 20 minutes. Meanwhile, dilute the labeled probe to 1 nanogram per microliter in hybridization buffer. For a negative control, use 10 times the concentration of non-labeled probe. Then, denature the probes by heating them at 90 degrees Celsius for 10 minutes, followed by quickly chilling them on ice.

Next, apply the same volume of probe dilution to each slide as proteinase K. Then, carefully cover slip and seal sections using nail polish. Now, heat the slides on a PCR machine block at 90 degrees Celsius for 10 minutes and transfer them to a 45 degrees Celsius humidified incubator overnight.

The next day, cool the slides down at 4 degrees Celsius for half an hour, and then carefully remove the cover slips. The tissues are fragile. Lastly, pass the slides through a series of SSC buffer at room temperature.

Wash the in situ hybridized slides for 5 minutes in MABT. Then, apply 50 to 400 microliters of blocking solution in 1% Tween 20 for 20 minutes. After the block incubation, apply 50 to 400 microliters of 1 to 1000 anti-DIG alkaline phosphatase antibody in blocking solution.

Let it bind for 90 minutes at 37 degrees Celsius. After the primary has been applied, wash the slides in MABT for 10 minutes. Then, for 5 minutes, put the slides in detection buffer with 1% Tween 20.

Now apply 50 to 400 microliters of 1 millimolar levamisole in detection buffer solution to each slide, and incubate them for 5 minutes to inactivate the endogenous alkaline phosphatase. Next, add 50 to 400 microliters of NBT/BCIP diluted in detection buffer at 1 to 100 to each slide, and let the slides incubate for 2 to 3 hours in a humidified chamber, based on experience.

Rinse the slides with DI water. Then, apply methyl green at 0.05% weight by volume and let the counterstain work on the tissue for 10 minutes at 37 degrees Celsius. Wash the slides with DI water again, and then dehydrate them with 100% ethanol, followed by a dip in xylene. Finally, apply 80 microliters of mounting media and cover slip the slides.