Visualization of Pseudomonas aeruginosa in Sputum Samples from Cystic Fibrosis Patients

الملخص

Source: Jackson, L., et al. Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients. J. Vis. Exp. (2020)

This video demonstrates the in situ visualization of Pseudomonas aeruginosa within chemically cleared cystic fibrosis sputum using fluorescent labeling and confocal microscopy to reveal bacterial localization and biofilm structure.

البروتوكول

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Hydrogel fluorescent in situ hybridization (FISH) protocol
   1. Remove the hydrogel samples from their storage solution using sterile tweezers and place the samples on a sterile surface (such as a glass slide or Petri dish).
   2. Using a sterile scalpel cut the hydrogels into ~ 1 mm thick slices.
   3. Place the 1 mm sections of hydrogel inside sterile 1.5 mL tubes.
   4. Prepare 1 mL the hybridization buffer (25% formamide, 0.9 M NaCl, 20 mM Tris-HCl [pH 7.6], 0.01% Sodium dodecyl sulphate (SDS), purified and deionized H2O) in a 1.5 mL tube.
   5. Add the fluorescently labeled PseaerA probe (150.7 nM) to the hybridization buffer and mix by inversion.
      NOTE: Formalin solution should be kept away from an open flame. The hybridization buffer can be prepared in advance and stored in aliquots at -20 °C.
   6. Add 200-500 µL of hybridization buffer to each ~1 mm section of hydrogel and ensure the entirety of the hydrogel sample is submerged.
   7. Allow the PseaerA probe to hybridize with the hydrogel samples by placing them in the dark for ~ 18-24 h, at 46 °C, without shaking.
   8. Decant the hybridization buffer into a waste collection container.
   9. Rinse samples once with filter sterilized wash buffer (337.5 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA (Ethylenediaminetetraacetic acid) [pH 7.2], 0.01% SDS, and purified and deionized H₂O) by adding 1 mL of wash buffer to each of the 1.5 mL tubes and then remove it.
      NOTE: The wash buffer can be made in advance and stored at room temperature (RT).
   10. Add 1 mL of fresh wash buffer to the tubes, then incubate the samples in the dark for 6 h at 48 °C, without shaking.
2. Hydrogel and Psl0096 antibody binding
   1. Sterilely remove the wash buffer using a 1 mL pipettor.
   2. Rinse the samples with a 2% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) solution by adding and removing 1 mL of the 2% BSA solution.
   3. Add 500 µL of the 2% BSA/PBS solution to the hydrogel samples to block non-specific protein binding. Then incubate the samples overnight, in the dark, at room temperature (RT), without shaking.
   4. Sterilely remove the blocking solution using a 1 mL pipette.
   5. Prepare the Psl0096-Texas Red antibody solution by diluting the antibody to a final concentration of 0.112 µg/mL in 500 µL of fresh 2% BSA/PBS.
   6. Add the 500 µL antibody solution to the hydrogel samples and incubate them at RT for 6 h, protected from light, without shaking.
3. DAPI (4′,6′-diamidino-2-phenylindole) staining
   1. Prepare the refractive index matching solution (RIMS) by adding 40 g of non-ionic density gradient medium, 30 µL of Tween20, 3 µg sodium azide, and 30 mL of PBS to a flask containing a magnetic stir bar. Stir the solution for 15 min on a magnetic stirrer, or until completely dissolved.
   2. Filter sterilize the solution into a 50 mL conical tube using a 10 mL syringe and a sterile 0.2 µm filter.
      NOTE: The solution can be stored at 4 °C for several months.
   3. Remove the Psl0096-Texas Red antibody solution with a sterile 1 mL pipette.
   4. Rinse the hydrogel samples by adding and then removing 1 mL of PBS.
   5. Incubate the hydrogel samples with 250 µL of RIMS solution and 10 µg/mL of DAPI at RT with gentle shaking, in the dark, overnight.
   6. Prior to confocal imaging mount the samples onto 0.9 mm or 1.7 mm perfusion chambers and seal with a glass coverslip.
      NOTE: After FISH and/or immunohistochemistry and before immersion in RIMS, fluorescent lectin stains can be applied if visualization of sputum mucous is desired.
4. Imaging
   1. Perform confocal laser scanning microscopy imaging using standard techniques at 25x, 40x, 63x, or 100x magnifications.

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
Coverwell perfusion chambers	Electron Microscopry Sciences	70326 -12/-14
PseaerA - GGTAACCGTCCCCCTTGC	Eurofins		Order Details: Product: Modified DNA Oligo; Name: PseaerA; Sequence: [Alexa488]GGTAACCGTCCCCCTTGC; Synthesis: 50 nmol; Purification: HPLC; Ship state: Full yield (dry)
Psl0096-Texas Red	Medimmune		The Psl0096-Texas red antibodies were a gift kindly provided by Medimmune and the company should be contacted for order inquiries.