High magnification in situ hybridization of LO from bioassay. High magnification example of an in situ hybridization reaction using a rhodamine-labeled ICE probe (red) together with an FITC-labeled LSNV probe (green) with lymphoid organ (LO) tissue of a P. monodon specimen from challenge test 1 that was positive by RT-PCR for both ICE and LSNV. This is a photomicrograph of a single confocal microscope image layer showing that the fluorescence distribution for ICE and LSNV is in the cell cytoplasm and not the nuclei. A lower magnification photomicrograph showing brighter fluorescence from multiple confocal microscope image layers is shown in Additional file 4. a) Phase contrast image; b) Image of LSNV fluorescence; c) Image of ICE fluorescence; d) Combined images with a magnified insert showing that the fluorescence distribution for the two signals is single (green or red) and combined (yellow). Note that the fluorescence is located in the cell cytoplasm and is most intense in the normal tubules of the LO and much less intense in the spheroids (i.e., the circular groups of cells surrounded by a ring of sheath cells that are most clearly visible in the combined image d). Negative control images from a normal shrimp specimen are shown in Additional file 3.