Anti mouse ly6g alexafluor647

The following antibodies were used for immunofluorescent labeling: anti-claudin-3 (Life Technologies), anti-rabbit IgG-FITC (Vector Laboratories, Burlingame, CA), anti-occludin-AlexaFluor⁴⁸⁸ (Life Technologies), anti-histone H2A/H4 (BWA3), anti-mouse IgG-TRITC (Jackson Immunoresearch), anti-mouse Ly6G-AlexaFluor⁶⁴⁷ (eBioscience, San Diego, CA), anti-mouse CD11c-AlexaFluor⁴⁸⁸ (eBioscience), anti-mouse surfactant A (Millipore), anti-rabbit IgG-AlexaFluor⁶⁴⁷ (Jackson Immunoresearch). Slides were mounted with ProLong Gold anti-fade reagent containing DAPI (Life Technologies). Digital monochromatic images were acquired on a Nikon A-1 confocal system with Nikon Elements software and pseudocolored. For quantitative analysis of histone-associated cells, greater than 200 histone-associated cells were analyzed per lung (n=3 mice) for cell type-specific labeling (neutrophil, macrophage, or type II alveolar epithelial cells). The percentage of histone-associated cells that were also labeled for the cell type-specific marker is displayed.

For confocal imaging of cells, PMNs and macrophages were plated on sterile 22 mm coverslips in 6-well plates for 1–2 hr to let cells adhere as best as possible. Then the activator (C5a or PMA) was added and incubated for 90 minutes at 37°C. Immunofluorescence staining followed. For studying uptake of histones, cells were incubated with FITC-conjugated histones or AF488-H1 up to 30 min at 37°C.
The following antibodies were used for immunofluorescent labeling: anti-mouse IgG-TRITC (Jackson Immunoresearch, West Grove, PA), anti-mouse IgG-AlexaFluor488 (Jackson Immunoresearch, West Grove, PA), and anti-mouse Ly6G-AlexaFluor647 (eBioscience, San Diego, CA). Slides were mounted with ProLong Gold anti-fade reagent containing DAPI (Life Technologies Grand Island, NY). Digital monochromatic images were acquired on a Nikon A-1 confocal system with Nikon Elements software.