Quantigene viewrna ish tissue 2 plex assay kit

Viral RNA was detected by in situ hybridisation using the QuantiGene® ViewRNA ISH Tissue 2-Plex Assay kit (Panomics/Affymetrix, Inc) according to the manufacturers׳ instruction. A mixture of three Probes sets targeting CnNPHV genomic RNA regions (nucleotides 840–2040, 3973–5010 and 7600–8634 of Accession no. JQ434001) corresponding to the core/E1 and E2 envelopes, NS3 helicase/NS4a and NS5b (RdRp) genes were used (Affymetrix). A Probe set for GAPDH RNA (Affymetrix, Accession no. NM_001003142) was employed as a positive control. Formalin-fixed, paraffin-embedded (FFPE) tissues sections (5+/−1 μm) were prepared on SuperFrost Plus slides. Sections were fixed for 2 h at 60 °C, deparaffinised, dehydrated, and permeabilized before the hybridisation steps and staining with Fast Red substrate (QG ViewRNA Chromogenic Signal Amplification kit (Panomics/Affymetrix, Inc.). The GAPDH positive control was detected with Fast Blue substrate (Panomics/Affymetrix, Inc.). Stained tissue sections were briefly treated with Meyer׳s haematoxylin (Sigma/Aldrich, UK) and dried before visualised by bright field light microscopy at 40× magnification.