Visualization of Bacteria in Bladder Biopsy Sections via Fluorescence In Situ Hybridization
Transcrição
Prepare a humidifying chamber for each probe by adding a soaked, crumpled, delicate task wipe and sterile water to the 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 with 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 slides into a vertical staining rack. Place the staining rack into an aluminum foil-wrapped coplin jar containing 100ml 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 counterstain by diluting a stock solution of Hoechst 33342 in wash buffer at a ratio of 1 to 1,000. 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 counterstain 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 cover-slipped 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 4 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 overcorrected. Acquire the image in all three channels.
