Biotinylated anti mouse igg

Retroperitoneal adipose tissue was included in Tissue-Tek^® medium, frozen in liquid nitrogen and 5 μm sections were obtained and adhered onto silanized slides. Slides were stained with HE. Evaluation of leukocyte infiltrate was also performed by immunohistochemistry. Slides were blocked with 3% BSA and incubated overnight with mouse anti-rat CD11b/c mouse antibody (BD Pharmingen ^™, 1:1000). Subsequently, the tissue was incubated with biotinylated anti-mouse IgG (Jackson ImmunoResearch, 1:200) secondary antibody for 2 hours, washed with 0.1M phosphate buffer and incubated with VectaStain ABC kit (Vector Laboratories, 1:100) for 2 hours. Detection of the antigen-antibody complex was performed through the chromogen 3,3'-diaminobenzidine (DAB) for 5 minutes at room temperature. Sections without the primary antibody (Cd11b/c) were used as negative control of the immunolabeling process. The qualitative evaluation of the slides was performed using photomicrographs captured by Zeiss Axiovert 40 microscope, with a 40x objective (Camera: Zeiss AxioCam ERc 5s).

ELISA plates were coated with 5 μg/ml NP₁₆ or NP₄ or with WNV envelope (E) protein overnight at 4°C in coating buffer containing 0.1 M sodium bicarbonate and 0.02% sodium azide at pH 9.6. Plates were washed three times with ELISA wash buffer (0.05% Tween 20 in PBS). Plates were blocked for 1 hour at room temperature with 2% BSA/PBS blocking buffer. Sera was then serially diluted in blocking buffer, plated and incubated for 1 hour at room temperature. Plates were washed three times followed by incubation with 1μg/ml biotinylated anti-mouse IgG (Jackson Immunoresearch), anti-IgG1^a-, or anti-IgG1^b (BD Biosciences) for 1 hour at room temperature. Wells were washed three times and then streptavidin-conjugated horseradish peroxidase (BD Biosciences) was added to each well and incubated for 1 hour at room temperature. Wells were washed three times with wash buffer and one time with PBS. Peroxidase activity was detected using tetramethylbenzidine (Dako) and quenched with 2 N H₂SO₄. Optical densities were measured at 450 nm. The end-point titer of each sample was calculated by a one-phase exponential decay curve and defined as the dilution that generated an OD₄₅₀ value that was 3 standard deviations above background. Prism software (GraphPad Software) was used to calculate the end-point titer.

ELISA plates (9018, Corning) were coated overnight at 4°C in 0.1 M sodium bicarbonate buffer, pH 9.5 containing 5 ug/mL of NP₁₆- or NP₄-BSA (Bioresearch Technologies). All other incubation steps were performed at room temperature for 1 hour. Wash steps were performed between each step using PBS + 0.05% Tween-20. Plates were blocked with PBS + 2% BSA followed by serial dilutions of serum. Serum was probed with 0.1 ug/mL of biotinylated anti-mouse IgG (715-065-151, Jackson ImmunoResearch Laboratories) and then detected with streptavidin conjugated horseradish peroxidase (554066, BD biosciences). Plates were developed using TMB (Dako, S1599) and neutralized with 2N H₂SO₄. Optical density (OD) values were measured at 450 nm. Serum endpoint titer was defined as the inverse dilution factor that is three standard deviations above background using one-phase decay measurements and Prism software (GraphPad Software).