Warp red

As previously described, representative liver, kidney, heart, brain, stomach, spleen, lung, uterus, urinary bladder, cecum, and small and large intestine samples were collected during necropsy and fixed in 10% buffered formalin for 24 h [18 (link)]. The tissues were subsequently transferred to 70% ethanol prior to being processed and embedded. Sections were stained with hematoxylin and eosin for histopathological evaluation. For immunohistochemistry (IHC), 3–4 μm sections were deparaffinized in CitriSolve (Decon Labs., King of Prussia, PA, USA) and rehydrated by processing through graded alcohols. Tissues were pretreated with Proteinase K (10 μg/mL) for 15 min. Endogenous IgG and non-specific background were blocked with Rodent Block M (Biocare Medical; Pacheco, CA, USA) for 20 min, followed by an alkaline phosphatase block (BLOXALL; Vector Laboratories, Burlingame, CA, USA) for 10 min. The primary antibody (PA1-7206; Thermo Fisher Scientific, Waltham, MA, USA) was incubated on the tissue sections at a dilution of 1:10,000 for 1 h at room temperature. Sections were sequentially incubated with a rabbit-on-rodent tissue alkaline phosphatase-based polymer and Warp Red (Biocare Medical, Pacheco, CA, USA). Tissues were counterstained with Tacha’s hematoxylin and mounted using EcoMount (Biocare Medical, Pacheco, CA, USA) [18 (link)].

Tissue sections, 3–4 μm in thickness, were deparaffinized in CitriSolve (Decon Labs., King of Prussia, PA) and rehydrated by processing them through graded alcohol solutions. Antigen unmasking was accomplished by digesting tissue in 10 μg/ml of protein kinase for 15 min at room temperature (Fisher Scientific, Waltham, MA). Endogenous enzymes and non-specific background were blocked with Background Punisher (Biocare Medical, Pacheco, CA), followed by BLOXALL (Vector Laboratories, Burlingame, CA). The primary antibody (NBP1–42140; Novus Biologicals, Centennial, CO) was incubated on the tissue sections at a dilution of 1:100 for 1 h at room temperature. Subsequently, sections were sequentially incubated with an alkaline phosphatase-based detection polymer kit (MACH 4; Biocare Medical), and Warp Red (Biocare Medical). Sections were counterstained with Tacha’s hematoxylin (Biocare Medical) and mounted using a permanent mounting medium (EcoMount; Biocare Medical).

Histopathology and detection of viral NP in the lung (immunohistochemistry) were performed at the Yerkes National Primate Research Center (Emory University, GA) according to procedures published previously (37 (link)). Briefly, the whole lungs were collected immediately after euthanization, fixed by submerging the tissues in 10% neutral buffered formalin, and embedded in paraffin. Five-micrometer sections of each lung lobe were taken and stained with hematoxylin and eosin (H&E). For immunohistochemistry staining, lung sections were stained with multisubtype mouse anti-NP antibody (1:1,000 dilution; MAB8251; Millipore, CA) by incubating them at room temperature for 1 h. The labeled tissue sections were stained with Mouse Mach2 (Biocare Medical), and all reactions were detected by development of the chromogen (Warp Red; Biocare Medical). Appropriate positive and negative controls were run in parallel. The nuclei were then counterstained using Gill's hematoxylin. Pathological scores were determined as described previously (38 (link)). Percent lung affected, severity of alveolitis and bronchiolitis/bronchitis, and extent of peribronchial and peribronchiolar/perivascular infiltrates were given the following scores: 0, no lesions; 1, ≤25%; 2, 25 to 50%; 3, >50%. Alveolar edema, hemorrhage, and type 2 pneumocyte hyperplasia were scored 0 if not present or 1 if present.