Biotinylated rabbit anti mouse ig

Kidney, liver, branchial arch and gills of the affected fish, which were extracted from the moribund fish, fixed in 10% formalin solution. Formalin-fixed tissues were embedded in paraffin wax and sections (4 μm) were stained with haematoxylin and eosin (HE). To detect IHNV in tissue sections, immunohistochemical procedures were performed as described by Kurath et al. (2016) (link). Briefly, the procedures included the use of 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS, pH 7.4) for blocking. The tissue samples were then washed with TBS. An IHNV-nucleoprotein-specific monoclonal antibody, Hyb 136–3 (Fregeneda-Grandes et al., 2009 (link)), provided by DTU, Denmark, was used as the primary antibody (1:3000 in TBS with 2.5% BSA). The tissues were incubated overnight at room temperature. The tissues were then washed three times with TBS and incubated with a secondary antibody (biotinylated rabbit anti-mouse Ig (DAKO Cytomation); 1:300). The tissue samples were incubated in streptavidin alkaline phosphatase complex (1:500; code RPN, 1234; Amersham Biosciences, Piscataway, NJ, USA) for 30 min at room temperature. The primary antigen-antibody reaction was visualized by adding fast red salt (F-2768; Sigma-Aldrich, St. Louis, MO, USA) to a fast red substrate solution (Polack and Noorden, 2003 ).