Division I.3.3 In-situ hybridisation (ISH)

Several ISH protocols have been developed and published.

Probe that targets the SSU of the rRNA gene complex shall be used because it has been validated against histology.

Sections of tissue that include gills, digestive gland, shall be fixed for at least 24 hours in Davidson's fixative followed by normal processing for paraffin histology. Sections of 5 μm shall be cut and placed on aminoalkylsilane-coated slides, which shall then be baked overnight in an oven at 40 °C. The sections shall be dewaxed by immersing in xylene for 10 minutes. This step shall be repeated once and then the solvent shall be eliminated by immersion in two successive absolute ethanol baths for 10 minutes each. The sections shall then be dehydrated by immersion in a graded ethanol series. The sections shall be treated with proteinase K (100 μg ml^{– 1}) in TE buffer (Tris [50 mM], EDTA [10 mM]), at 37 °C for 30 minutes. Slides shall be dehydrated by immersion in a graded ethanol series and then air dried. Sections shall be incubated with 100 μl of hybridisation buffer (4 × SSC [standard saline citrate], 50 % formamide, 1 × Denhardt's solution, 250 μg ml^{– 1} yeast tRNA, 10 % dextran sulphate) containing 10 ng (1 μl of the PCR reactants prepared as described in point I.3.2 using primers CCG-GTG-CCA-GGT-ATA-TCT-CG and TTC-GGG-TGG-TCT-TGA-AAG-GC) of the digoxigenin-labelled probe. Sections shall be covered with in-situ plastic cover-slips and placed on a heating block at 95 °C for five minutes. Slides shall then be cooled on ice for one minute before overnight hybridisation at 42 °C in a humid chamber. Sections shall be washed twice for five minutes in 2 × SSC at room temperature, and once for 10 minutes in 0,4 × SSC at 42 °C. The detection steps shall be performed according to the manufacturer's instructions. The slides shall then be rinsed in sterile distilled water (dH2O). The sections shall be counter-stained with Bismarck Brown Yellow, rinsed in dH2O, and cover-slips shall be applied using an aqueous mounting medium.

Positive and negative controls shall be sections from known infected and non-infected hosts respectively.

A positive result shall be demonstrated by the purple-black labelling of M. refringens cells within known target tissues, with all negative controls being negative and all positive controls being positive.