5.6.2. Performing fluorescence in situ hybridization

  1. Sections should be rehydrated and prepared for hybridization as follows in order to improve permeability:
    1.1. Wash slides with xylene 3 x 10 min.
    1.2. Wash slides with 100 % ethanol 3 x 3 min.
    1.3. Wash slides with 90 % ethanol 2 x 3 min.
    1.4. Wash slides with 80 % ethanol 2 x 3 min.
    1.5. Wash slides with 70 % ethanol 1 x 3 min.
    1.6. Wash slides with 50 % ethanol 2 x 3 min.
    1.7. Wash slides with double distilled water (DEPC) 2 x 3 min.
    1.8. Incubate slides with 1 mg/ml Proteinase K in 0.2M Tris-HCl (pH 7.9) for 5 min at 37°C in a humid chamber.
    1.9. Incubate slides with 1 mg/ml lysozyme in DEPC double-destilled water for 15 min at 37°C in a humid chamber.
    1.10. Wash slides three times with 1X PBS.
  2. Incubate slides with 100 ng of each probe (specific bacterial 16S rRNA-probe and universal eukaryotic 18S rRNA-probe) diluted in 20 µl of FISH-hybridization buffer (20 % (v/v) deionized formamide, 0.9 M NaCl, 20 mM Tris-HCl pH 7.9, 0.01% (m/v) SDS).
  3. Cover with slip and transfer to a Corning chamber.
  4. Dispense double-distilled water into the cavities of the chamber and close it.
  5. Incubate in a humid chamber at 46°C from 4 h to overnight.
  6. Carefully remove cover slip in 1X PBS.
  7. Wash slides three times with 1X PBS.
  8. Add 50 µl of DAPI solution (1 µg/ml in methanol) to each slide.
  9. Cover again.
  10. Incubate at RT for 10 min.
  11. Remove cover slip in 1X PBS and wash slides three times with 1X PBS.
  12. Mount slides with antifade reagent.