Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

Here we present a robust protocol for the detection of bacteria within biopsied bladder tissue, which has broad applications to many tissue types where bacteria are suspected to be present. The main advantage of this technique is that it's unbiased. It allows the researcher to detect bacteria within tissue without a priori knowledge of which bacterial species is present.

This technique may help to determine if bacteria have invaded the bladder walls of women with recurrent urinary tract infection and inform therapeutic choices such as the selection of tissue-penetrating antibiotics. While designed for bladder tissues, this technique can be broadly applied to other tissues with a resident bacterial populations with minimal optimization. Individuals who are new to this technique may struggle with coverslip placement and imaging.

Focus on optimizing coverslip placement to reduce bubbles and practice imaging on non-precious samples. Helping to demonstrate this procedure will be Jashkaran Gadhvi, a graduate student in my lab. To begin this procedure, clean a empty fume hood or appropriately fitted biosafety cabinet with 70%ethanol.

Prepare all of the necessary buffers and reagents as outlined in the text protocol. Next, clean five Coplin jars with 70%ethanol and let the jars dry. When the jars are dry, label them as shown here and fill them with 100 milliliters of the appropriate solution.

In the fume hood, place two slides per biopsy into a vertical slide rack. Place the vertical slide rack into the Xylenes 1 Coplin jar for 10 minutes. After this, remove the slide rack from the jar and blot the bottom on a paper towel to remove excess xylenes.

Place the rack into the Xylenes 2 jar for 10 minutes. And then rehydrate the de-paraffinized tissue sections in successive ethanol washes for 10 minutes each. When the washes are complete, blot the bottom of the slide rack on paper towels to remove excess ethanol and place the rack in the Coplin jar containing 100 milliliters of filter sterilized, double distilled water for 10 minutes.

During this wash, dilute the probes to 10 nanomolar in hybridization buffer to create the staining solution, making sure to prepare 150 microliters of staining solution per slide. Prepare a humidifying chamber for each probe by adding a soaked, crumpled delicate task wipe and sterile water to a reservoir of a P1000 tip box. Place the tip holder cartridge on top as this is where the slides will sit.

When the final wash is complete, remove the slide from the slide rack and place them onto a fresh paper towel, tissue-side up. Use a delicate task wipe to dry the slide, being careful to only gently dab near but not on the biopsy section to wick away the water. Using a hydrophobic pen, draw a border around the biopsy section and place the slide tissue side up in the humidifying chamber.

Next, place the humidifying chamber into an incubator set to 50 degrees Celsius. Pipette between 50 and 150 microliters of the staining solution directly on top of the tissue so that the rectangle made by the hydrophobic border around the tissue is filled and close the box gently. Incubate overnight at 50 degrees Celsius in the dark.

If the incubator has a window, cover it wit aluminum foil to create a dark environment. The next morning, remove the slides from the humidifying chambers and quickly wick away any remaining hybridization solution with a delicate task wipe. Then place the sides into a vertical staining rack.

Place the staining rack into an aluminum foil wrapped Coplin jar containing 100 milliliters of the pre-warmed wash buffer for 10 minutes. Repeat this wash step two more times with fresh wash buffer in new Coplin jars. During these wash steps, prepare the counter-stain by diluting a stock solution of Hoechst 33342 in wash buffer, at a ratio of one to 1000.

To the same tube, add Alexa-555 wheat germ agglutinin to a final concentration of five micrograms per milliliter and Alexa-555 Phalloidin to a final concentration of 33 nanomolar. Store in the dark until ready to use. When the final wash is complete, remove the slides from the Coplin jar and gently wick away any excess wash buffer with a delicate task wipe.

Place the slides tissue-side up on a paper towel and add between 50 and 150 microliters of counter-stain directly on top of the tissue so that the hydrophobic border is filled, but not overflowing. Cover up to four slides with a cryobox-top and incubate at room temperature for 10 minutes. After this, place the slides back into the staining rack and wash them twice more in Coplin jars containing fresh wash buffer, with each wash lasting 10 minutes.

Then thoroughly dry the slides and place them tissue-side up on a paper towel under a cryobox-top. Squeeze one drop of mounting media directly on top of the tissue and gently place an appropriately sized coverslip on top. Gently press out any bubbles and allow the coverslip slides to cure overnight in the dark.

The next day, seal the edges of the coverslips to the slide with a light coat of clear nail polish and let dry in the dark for 10 minutes, before storing them in the dark at four degrees Celsius. When ready to image the stained biopsies, switch on the confocal microscope and the software associated with the microscope. Start with a FISH positive slide and switch to the computer visualization mode.

Select the channels for 405, 488 and 555. Set the pinhole using the longest wavelength channel, which in this case is 555. Then find the current focal plane for visualization of labeled bacteria in the 488 channel.

Without changing the focal plane, set the gain, laser power, and offset for each channel such that the signal is not saturated and the background is not over-corrected. Acquire the image in all three channels. In this study, tissue associated bacteria are detected in human bladder biopsies by 16S rRNA FISH.

This protocol has been optimized for the unbiased detection of bacteria associated with the bladder mucosa in paraffin-embedded bladder biopsy sections. Shown here are representative confocal micrographs from an experiment using this protocol on sections of bladder biopsies obtained from women with recurrent urinary tract infections. Two serial sections are hybridized with either the universal 16S rRNA or scramble probes.

Images ar taken from the same region of the tissue and bacteria are cleanly visible in the tissue hybridized with the 16S rRNA probes, but are not with the scramble probe. False positive results are also possible. In this representative false positive, a signal corresponding to autofluorescent collagen or elastin is detected in the 405 and 488 channels in both the 16S rRNA and scramble probe hybridized biopsy sections, highlighting the importance of always using a scramble probe control.

To prevent photo-bleaching, minimize exposure of tissue to light after probes are applied. While this technique allows for the unbiased detection of bacteria in human tissues, it does not give species specific information. Multiplex-FISH with taxonomy specific probes could be used to identify the genus or species present.

Before this technique was developed, it had not been previously demonstrated that bacteria could invade the bladder wall of human recurrent UTI patients. We are now using genus and species specific probes to determine which bacterial species are present. Xylenes should be used in a fume hood due to their toxicity.

Care should be taken not to look directly into the laser during imaging. The laser should be turned off while positioning your slide under the objective.