5.6. Fluorescent in situ hybridization for the detection of Paenibacillus larvae

Recently, Yue and collaborators (Yue et al., 2008) described a P. larvae specific method based on a previously described general technique of fluorescence in situ hybridization (FISH) (Moter et al., 1998). FISH methods are based on specific binding of fluorescent-labelled oligonucleotide probes to complementary sequences in fixed and permeabilized sections (Itzkovitz and van Oudenaarden, 2011). This approach provides a highly specific method to visualize P. larvae in infected larval histological sections, thereby allowing disease monitoring and observation of the life cycle of the bacterium inside the host. P. larvae FISH methodology uses a specific complementary P. larvae 16S rRNA targeted oligonucleotide probe coupled with fluorescein isothiocyanate (FITC), that specifically binds to P. larvae rRNA. Since bacterial cells are filled with ribosomes, this technique allows the ”staining“ of the vegetative bacteria. In order to simultaneously visualize the cytoplasm of larval cells, cyanine Cy3-labeled oligonucleotides universally detecting conserved 18S rRNA eukaryotic sequences are used. Finally, 4',6-diamidino-2-phenylindole (DAPI), a fluorescent dye that binds A-T rich regions of DNA, is employed to visualize cell nuclei.

         The following protocol provides a method to fix and embed larval tissues, in order to obtain histological sections, as well as for further processing these sections by fluorescence in situ hybridization.

5.6.2. Performing fluorescence in situ hybridization