For the histopathological and immunohistochemical examination, tissue samples collected from the brain, trachea, lung, heart, liver, spleen, proventriculus, pancreas, duodenum, ileum, and kidney were fixed in 10% neutral buffered formalin. The tissues were then routinely processed and embedded in paraffin. For histopathology, the paraffin blocks were cut on microtome into 4 µm-thick sections, which were placed on standard glass slides and stained with haematoxylin and eosin (H&E). For immunohistochemistry, the tissue sections cut from the paraffin blocks were placed onto Superfrost glass slides (Menzel–Glaser, Braunschweig, Germany) and incubated in 37 °C overnight. Next, the sections were deparaffinized, rehydrated in descending ethanol concentrations, and subjected to endogenous peroxidase blocking using 3% solution of H2O2 (30%) in methanol for 10 min, followed by epitope unmasking using protease K (DAKO, Glostrup, Denmark) for 15 min at room temperature. For the detection of viral antigen, anti-influenza A nucleoprotein monoclonal antibody (HYB 340-05, Statens Serum Institute, København, Denmark) was used (dilution 1:1000, 2 h). The antibody detection was performed using a Dako REAL EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse (K5007, DAKO, Glostrup, Denmark). The sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted. To confirm the specificity of the staining, sections incubated with PBS instead of the primary antibody were used. The tissues were examined under a light microscope (Axiolab, Zeiss, Jena, Germany) for evaluation of histopathological lesions in the H&E-stained sections and the detection of the immuno labelling of the viral antigen in the IHC-stained ones. For an assessment of histopathological lesions, the semiquantitative scoring system proposed by Landnann et al. [22 (link)] was applied.
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