9.4. FISH-analysis of tissue sections and fixed insect cells

  1. Wash the slides (tissue sections and fixed insect cells) twice in 1xPBS.
  2. For further processing, transfer each slide into a 10 ml dish.
  3. Add 10 ml of 1 µg ml-1 Proteinase K in Proteinase K-buffer (0.2 M Tris-HCl, pH 7.5).
  4. Transfer the dish into a humid chamber (Eppendorf, Thermostat Plus) for 5 min at 37°C.
  5. Remove the Prot K and wash each slide with 10 ml of 1xPBS.
  6. For post-fixation add 10 ml of 4% formalin (Roth).
  7. Incubate at RT for 20 min.
  8. Aspirate the formalin.
  9. Remove remaining solution by washing the slides three times with 1xPBS-buffer.
  10. Prepare hybridization buffer:
    200 µl of 100% formamide,
    180 µl 5 M NaCl,
    20 µl 1 M Tris/HCl,
    1 µl 10 % SDS,
    599 µl DEPC-H2O, pre-warm to 46°C in a heating block.
  11. Add the probes to 37.5 µl pre-warmed (46°C) hybridization buffer:
    11.1. 7.5 µl species-specific probe annealing to a region of the 16S rRNA or another species-specific genomic region of the pathogen to be detected labelled with fluorescein isothiocyanate-FITC with a final concentration of 15 ng µl-1.
    Sequence of Nosema spp.-probes: Gisder et al. (2011); sequence of DWV-probe: Möckel et al. (2011); sequence of P. larvae-probe: Yue et al. (2008)
    11.2. 5 µl Euk516-probe (5‘-ACCAGACTTGCCCTCC-3‘, universally detecting eukaryotic ribosomes by hybridizing to a universal conserved sequence of the eukaryotic 18S rRNA) labelled with sulforhodamine 101 acid chloride-Texas Red® with a final concentration of 10 ng µl-1.
  12. Continue incubation in the heating block which is now covered with a lid (i.e. incubation at 46°C in the dark).
  13. Cover the slides with LifterSlips (VWR).
  14. Pipette 50 µl of hybridization buffer to each slide (tissue sections or fixed cells).
  15. Transfer the slide into a hybridization chamber (Corning, Corning chamber), drop 15 µl H2O into the given wells to preserve the humidity.
  16. Close the chamber tightly.
  17. Put the corning chamber in a 46°C water bath for overnight hybridization.
  18. Open the hybridization chamber and remove the cover slips in 1xPBS.
  19. Wash the slides three times with 1xPBS.
  20. Let them air dry.
  21. Stain the nuclei with 50 µl 4′, 6-Diamidin-2-phenylindol- (DAPI, 1 µg ml-1 in 99% methanol) solution for 10 min in the dark.
  22. Wash the slides again three times with 1xPBS.
  23. Let them air dry.
  24. Cover the slides with the ProLong Gold antifade reagent (Invitrogen) and a cover slip (Roth).
  25. Analyse the tissue sections and the cells under an inverse fluorescence microscope (e.g. Nikon, Ti-Eclipse) at 100-fold and 600-fold magnification using consecutively a FITC-, TexasRed- and DAPI-filter.