Necropsies for all experiments were performed according to standard procedures under biosafety level-3 (BSL-3) conditions. Varying between individual experiments, specimens of brain, skin, nasal cavity, oral cavity, trachea, lung, air sacs, heart, thymus, glandular stomach, gizzard, duodenum, jejunum, caecum, liver, kidney, spleen, pancreas, adrenal gland, bursa fabricii, ovary/testis, and chorioallantoic membrane were taken. Samples were fixed in 4% neutral-buffered formaldehyde for ≥7 days, trimmed, processed, and embedded in paraffin wax. In total, 2–5 µm microtome slices were mounted on glass slides and stained with hematoxylin and eosin.
Immunohistochemical examination was performed on formaldehyde-fixed and paraffin-embedded (FFPE) tissue samples with the avidin-biotin-peroxidase complex method (Vector Laboratories, Burlingame, CA, USA) using a polyclonal rabbit anti-influenza A-nucleoprotein (NP) antibody [33 (link),47 (link),48 (link)] or monoclonal murine anti-influenza A-matrixprotein (MP) antibody [41 (link)] with 3-amino-9-ethyl-carbazol as chromogen and hematoxylin counterstain in individual experiments (see Table S1). Validated positive and negative archival tissues were used as controls, and the specific antibody was replaced by, e.g., Tris-Buffered Saline (TBS) [41 (link)] or rabbit serum [42 (link)].
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