Rnascope 2.5 red assay kit

To demonstrate viral genome in tissue sections, colorimetric in situ hybridization (ISH) was performed on 5-μm-thick sections of formalin-fixed, paraffin-embedded tissues on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) using the RNAscope 2.5 Red assay kit (Cat #322360, Advanced Cell Diagnostics, Inc., Hayward, CA). We designed V-UaCirV, (ACD Cat #555001) as 26 ZZ-paired probe sets targeting a 1409 nt segment of the viral genome corresponding to nucleotide positions 478–1887 of the reference sequence (GenBank accession MN371255). Selected tissues included livers and spleens, as these are established sites of circoviral distribution in related species, as well as brains in order to evaluate the possible association with neurologic disease in these cases. Each 5-μm-thick tissue section was pretreated with heat and protease prior to probe hybridization for 2 hours at 40°C. Negative controls used for validation of signal included an unrelated (GC-content matched) probe on serial sections. Slides were counterstained with hematoxylin and mounted with EcoMount (Biocare Medical, Concord, CA).

The detection of CaHV-1 was performed using the RNAscope 2.5 Red assay kit (Advanced Cell Diagnostics [ACD] Cat No. 322360, Hayward, CA, USA). The RNAscope probe was designed by Advanced Cell Diagnostics to detect the mRNA expression of the Canine herpesvirus thymidine kinase gene (V-CHV-TK) (Cat No. 457121). Deparaffinized and dehydrated 5 µm thick tissue sections were incubated in hydrogen peroxide for 10 min and soon after in a target retrieval buffer (ACD) at 98 °C/100 °C using a hot plate for 15 min. Tissue sections were then treated with a Protease Plus reagent (ACD) in a hybridization oven at 40 °C for 25 min (ACD, Hayward, CA, USA) and subsequently incubated at 40 °C with the target probe in the same hybridization oven for 2 h. A probe targeting the PPIB gene (Cyclophilin B) and a probe targeting the DapB gene (Bacillus subtilis strain SMY) were used as positive and negative controls, respectively. A series of target-specific signal-amplification steps were conducted for both assay kits and Fast Red signal-amplification system was then hybridized to the target probes. Slides were counterstained with hematoxylin and cover slipped using Ecomount (Biocare Medical, Concord, CA, USA). The sections were examined under a light microscope (Leica DM4000B).