9.4. FISH-analysis of tissue sections and fixed insect cells
- Wash the slides (tissue sections and fixed insect cells) twice in 1xPBS.
- For further processing, transfer each slide into a 10 ml dish.
- Add 10 ml of 1 µg ml-1 Proteinase K in Proteinase K-buffer (0.2 M Tris-HCl, pH 7.5).
- Transfer the dish into a humid chamber (Eppendorf, Thermostat Plus) for 5 min at 37°C.
- Remove the Prot K and wash each slide with 10 ml of 1xPBS.
- For post-fixation add 10 ml of 4% formalin (Roth).
- Incubate at RT for 20 min.
- Aspirate the formalin.
- Remove remaining solution by washing the slides three times with 1xPBS-buffer.
- 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.
- 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.
- Continue incubation in the heating block which is now covered with a lid (i.e. incubation at 46°C in the dark).
- Cover the slides with LifterSlips (VWR).
- Pipette 50 µl of hybridization buffer to each slide (tissue sections or fixed cells).
- Transfer the slide into a hybridization chamber (Corning, Corning chamber), drop 15 µl H2O into the given wells to preserve the humidity.
- Close the chamber tightly.
- Put the corning chamber in a 46°C water bath for overnight hybridization.
- Open the hybridization chamber and remove the cover slips in 1xPBS.
- Wash the slides three times with 1xPBS.
- Let them air dry.
- 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.
- Wash the slides again three times with 1xPBS.
- Let them air dry.
- Cover the slides with the ProLong Gold antifade reagent (Invitrogen) and a cover slip (Roth).
- 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.