Rat igg2b

Immunohistochemistry for macrophages was performed on frozen serial sections (adjacent to those stained with Oil Red O) of the ascending aorta using the MicroProbe system as described previously using rat anti-mouse CD68 (Bio-Rad, Cat# MCA1957), Rat IgG2b (BD PharMingen, Cat# 559478), and biotinylated rabbit anti-rat IgG (Vector, Cat# BA-4001) antibodies^{59 (link)}. Immunoreactivity was visualized with red chromogen 3-amino-9-ethylcarbazole (AEC) (Vector). Macrophage content was quantified in the first serial section of the aortic root from the internal elastic lamina to the luminal edge using Image-Pro 7.0 software by 2 researchers as described above. The area of CD68 staining was expressed as a percentage of atherosclerotic lesion area.

Bronchoalveolar lavage fluid cells were collected (from 4–6 mice per group) and the pooled cells were FcγR blocked using 2.4G2 antibody (ATCC) and stained with combinations of the following mouse conjugated mAb: allophycocyanin (APC) or FITC anti-CD3, APC/Cy7 anti-CD4, PE anti-CD8a, APC/Cy7 anti-Ly-6G/Ly6C (Gr-1), PE, FITC or Brilliant Violet 421 anti-CD11b, APC or PE anti-F4/80 (all purchased from BioLegend, San Diego, CA, United States). In addition, PE anti-Siglec-F (BD Biosciences) was used to stain eosinophils. Flow cytometric acquisition was performed on a FACSAria II (BD Biosciences) by 4-color analysis using FACSDiVa software and FlowJo, with a minimum of 50,000 live, single-cell events per sample collected. Cells were live gated based on Forward versus Side Scatter properties. In addition, isotype controls were used to control for non-specific background antibody binding. To this end, cells were stained using conjugated (APC, FITC, PE, APC/Cy7, or Brilliant Violet 421) control rat IgG2a, rat IgG2b, and hamster IgG1 antibody (obtained from Becton Dickinson).

For neutralization experiments, rgRSV was incubated with increasing concentrations of heparan sulfate (Sigma-Aldrich, St. Louis, MO), protein A-purified mAb L9 [35 (link)] or mAb 131-2g [36 (link)] (gift from Dr. L.J. Anderson, Emory University School of Medicine, Atlanta, GA), compared to the appropriate mouse isotype controls, IgG_2a or IgG₁ (R&D Systems, Minneapolis, MN). RSV isolate 2–20 or B1 were incubated with 5 μg/ml mAb 131-2g. All mAbs were diluted in DMEM 10% FBS and incubated with virus for 30 min at room temperature prior to inoculation of HeLa and HAE cultures. For anti-CX3CR1 blockade experiments, cells were incubated with 25 μg/ml CX3CR1 specific rat mAb (clone 2A9-1) (MBL International Corporation, Woburn, MA) or rat IgG_2b (Becton Dickinson, East Rutherford, NJ) for 30 min prior to inoculation. HeLa and HAE cultures were inoculated with ~200 PFU at 37°C. The inoculum was removed 2 hr later and cultures rinsed 3 times with PBS. At 24 hr post inoculation, cultures were fixed, permeabilized and incubated with an FITC-labeled anti-RSV polyclonal antibody (Virostat, Portland, ME). Infected (green from GFP or FITC) cells were detected and counted on an EVOS FL Cell Imaging System (Life Technologies, Carlsbad, CA).