Heterogeneous pathological outcomes after experimental pH1N1 influenza infection in ferrets correlate with viral replication and host immune responses in the lung

Vidaña, Beatriz; Martínez, Jorge; Martínez-Orellana, Pamela; García Migura, Lourdes; Montoya, María; Martorell, Jaime; Majó, Natàlia

Cytotoxic lymphocytes and natural killer (NK) cells were detected using the anti-CD8a (Sino Biological, Polyclonal Rabbit anti-ferret CD8a Antibody, ref 60001-RPO2, China) polyclonal antibody. Briefly, tissue sections were deparaffinised with xylene and rehydrated through graded concentrations of alcohol. Endogenous peroxidase activity was blocked by incubation with 3% H 2 O 2 in methanol for 30 min. Tissue sections were rinsed in PBS and immersed in Retrieval Solution (Dako, Target Retrieval solution 10x concentrate, n°S1699) for antigen retrieval. Later, the slides were blocked with 2% bovine serum albumin (85040C, Sigma-Aldrich Química, S.A., Spain) for one hour at RT and incubated with the primary antibody overnight at 4°C at a dilution of 0.5 μg/mL. Next, a polymer-based non-avidin-biotin peroxidase system (Dako EnVision® + System, Peroxidase-HRP, Dako, Denmark) was applied directly to the slides and incubated for 30 min at RT. The reaction was developed with DAB (Sigma-Aldrich, Madrid, Spain) at RT, followed by counterstaining with Mayer's haematoxylin. Ferret lymph node sections were used as positive controls. The same sections in which the specific primary antibodies were substituted with PBS were used as negative controls. Semiquantitative assessments of the inflammatory cells detected in the lungs were also performed. The cells that were positive in the IHC for the anti-lysozyme (macrophages and neutrophils), anti-CD3 (T cells), anti-CD8a (cytotoxic T cells and NK cells) and anti-CD20 (B cells) antibodies were quantified for every animal and antibody. Positive cells in 6 arbitrarily chosen 40× objective fields in alveolar areas and 5 arbitrarily chosen 40× objective fields in bronchi or bronchiolar areas were counted separately for each lobe (cranial, medial and caudal) of every animal. The means of the total cell counts per field across the three lung lobes were then calculated for each animal and antibody.