Nuclear fast red

The presence of C. perfringens harboring netB gene in
tissue sections was detected by ISH. The ISH was performed as described previously [6 (link)] with some modifications. Briefly, deparaffinized
sections were treated with 0.2 N HCl at room temperature for 20 min and then digested with
proteinase K (Sigma-Aldrich Japan Inc., Tokyo, Japan) at 37°C for 15 min. The sections
were then incubated overnight at 42°C in standard hybridization buffer together with the
netB-specific probe. After the washing and blocking steps, the sections
were incubated with anti-DIG antibody conjugated with alkaline phosphatase diluted 1/200
in blocking reagent (Boehringer Mannheim, Co., Ltd., Tokyo, Japan) at room temperate for 1
hr and then detected by BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue
tetrazolium chloride). Color was allowed to develop for 10 min in the dark. The sections
were counterstained with Nuclear Fast Red (Roche Diagnostics GmbH) and systematically
viewed under a light microscope. No background hybridization was seen when replicate
tissue sections were incubated with an unrelated digoxigenin-labeled probe (PCV2-specific
probe) [6 (link)] or when matched tissues from unaffected
chickens were incubated with a netB gene-specific probe.

ISH assay was performed using miRCURY locked nucleic acid (LNA) detection probe for miRNA-532-5p (Vedbaek, Denmark). Five hundred nanomoles per liter of digoxigenin double-labeled LNA-modified miR-532-5p probe was added to the hybridization solution and hybridized according to the instructions. After the incubation of slides with 4-nitroblue-tetrazolium and incubation with nuclear fast red (Roche Applied Science, Indianapolis, IN, USA), the images were captured under inverted microscope (Olympus, Tokyo, Japan).

Biopsy specimens were fixed in formalin, dehydrated, embedded in paraffin, and sectioned with routine methods. The INFORM EBER probe (Ventana Medical Systems, Tucson, AZ, USA) was used for EBER‐ISH. Slides were stained on an automated stainer (Benchmark XT; Ventana Medical Systems), and for visualization the ISH iView Blue Detection Kit (Ventana Medical Systems) with alkaline phosphatase and NBT/BCIP substrate was used, with Nuclear Fast Red (Ventana Medical Systems) for contrast. Specimens in which nuclear expression of EBER was observed in 20% or more of the malignant cells were considered EBER‐positive.15