Metal enhancer dab stain

Lung tissues were fixed with 4% formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE). Immunostaining was performed using a four-clone blend of monoclonal antibodies against the RSV P, F, and N proteins (AdB Serotec, UK) and anti-mouse IgG conjugated with HRP (Dako North America, Inc.)[17 (link)]. Lung tissue specimens were stained using the Peroxidase Stain DAB Kit and Metal Enhancer DAB stain (Nacalai Tesque, Inc., Kyoto, Japan).
As for the scoring of histological findings, six random fields per group were scored for histopathology using the following criteria: 1, swelling of alveolar walls; 1, destruction of bronchial epithelial cells; 1, peribronchial infiltration of inflammatory cells; 2, mucus as a production cause of occlusion in the bronchial space; and 1, bleeding. Microscopic images were taken with a Life Technologies EVOS XL Core light microscope at 40× magnification.

NT against RSV was performed with the 50% plaque reduction assay using the Long strain, as previously reported [16 (link)]. Briefly, serum samples were serially diluted two-fold, starting from a 1:10 dilution, and then mixed with an equal volume of RSV subgroup A (100 PFU) at room temperature for one hour. In the present study, NT antibody titers were examined not only against the Long strain, but also against wild strains. Serum samples were serially diluted two-fold, starting from a 1:5 dilution, and then mixed with an equal volume of RSV subgroup B (100 TCID₅₀) at room temperature for one hour. The mixture was incubated on Vero cells at 37°C for 6 days in 5% CO₂. These cells were fixed with 99.5% ethanol for 10 minutes and washed with PBS. Polyclonal antibodies against RSV (Abcam, Cambridge, UK) were diluted at 1:1,000 and incubated at room temperature for two hours. The antibodies were removed and cells were washed with PBS. Anti-goat IgG-HRP (Santa Cruz Biotechnology, Inc.), diluted at 1:1,000, was then added, and the plate was incubated at room temperature for 40 minutes. The plate was washed with PBS, and Vero cells were stained using the Peroxidase Stain DAB Kit with a Metal Enhancer DAB stain (NACALAI TESQUE, INC. Kyoto, Japan). NT antibody titers were expressed as the reciprocal of the highest dilutions that inhibited the appearance of the CPE of RSV.