JoVE 실험 백과사전
미생물학
0 조회수 • 3:43 분 • July 1st, 2026
Take a tube containing solidified hydrogel embedded with cystic fibrosis patient-derived sputum.
The sputum contains infection-associated Pseudomonas aeruginosa, a pathogenic bacterium.
Add a hybridization buffer containing green-fluorescent probes and incubate to allow the probes to bind selectively to bacterial ribosomal RNA.
Rinse with wash buffer to remove unbound probes.
Add fresh wash buffer and incubate to stabilize probe binding.
Remove the buffer, then add a blocking buffer to prevent nonspecific interactions.
Remove the blocking buffer. Introduce a solution containing red-fluorescent antibodies, and incubate to allow the antibodies to target the bacterial exopolysaccharide.
Rinse with a buffer to remove unbound antibodies, then stain with a DNA-binding dye to label DNA.
Mount the stained hydrogel onto a perfusion chamber with a coverslip.
Under a confocal microscope, observe green-fluorescently labeled bacteria surrounded by red-fluorescently labeled exopolysaccharide, allowing clear visualization of bacteria within the sputum.
Remove the hydrogel samples from their storage solution and place the samples on a sterile surface. Use a sterile scalpel to cut the hydrogels into approximately one millimeter thick slices, and place the sections into 1.5 milliliter tubes. Prepare one milliliter of hybridization buffer in a 1.5 milliliter tube, then add the fluorescently labeled PseaerA probe, and mix by inversion.
Add 200 to 500 microliters of hybridization buffer to each section, making sure that the entirety of the hydrogel sample is submerged. Allow the PseaerA probe to hybridize with the hydrogel samples by placing them in the dark for approximately 18 to 24 hours at 46 degrees Celsius without shaking. Then, add one milliliter of fresh wash buffer to the tubes, and incubate them in the dark for six hours at 48 degrees Celsius. Add 500 microliters of the 2% BSA in PBS to the hydrogel samples to block non-specific protein binding. Then, incubate the samples overnight in the dark.
On the next day, prepare the Psl0096-Texas Red antibody solution by diluting the antibody to a final concentration of 0.112 micrograms per milliliter in 500 microliters of fresh 2% BSA in PBS. Add the antibody solution to the hydrogel samples, and incubate them in the dark for six hours.
Next, stain the hydrogel samples with DAPI by incubating them with 250 microliters of RIMS solution and 10 micrograms per milliliter of DAPI overnight in the dark with gentle shaking.
After staining, mount the samples onto 0.9 or 1.7 millimeter perfusion chambers and seal them with a glass coverslip. Perform confocal laser scanning microscopy with 25, 40, 63, or 100 times magnifications.