5.6.2. Performing fluorescence in situ hybridization
- 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.
- 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).
- Cover with slip and transfer to a Corning chamber.
- Dispense double-distilled water into the cavities of the chamber and close it.
- Incubate in a humid chamber at 46°C from 4 h to overnight.
- Carefully remove cover slip in 1X PBS.
- Wash slides three times with 1X PBS.
- Add 50 µl of DAPI solution (1 µg/ml in methanol) to each slide.
- Cover again.
- Incubate at RT for 10 min.
- Remove cover slip in 1X PBS and wash slides three times with 1X PBS.
- Mount slides with antifade reagent.