Immunocytochemical stainings were performed as described previously (Schröder et al., 2010 (link)). Cells grown on coverslips were fixed with 4% (wt/vol) paraformaldehyde. Lysosomal compartments were visualized with the 2D5 monoclonal LAMP-2 antibody (Radons et al., 1992 (link)). Alexa 488- and 594-conjugated secondary antibodies were purchased from Molecular Probes. Nuclei were stained with DAPI (Sigma-Aldrich), which was included in the embedding medium at a concentration of 1 µg/ml. Images were acquired with an Olympus FV1000 confocal laser scanning microscope and further processed with Olympus Fluoview software and Adobe Photoshop software.
Fluoview software
Manufactured by Adobe
Fluoview software is a digital imaging and analysis tool designed for use with fluorescence microscopy systems. It provides core functionalities for controlling and managing the acquisition of fluorescence images, as well as basic tools for image processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Alexa 488 and 594 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Sytox orange, by Thermo Fisher Scientific (1 mentions)
Fluoview software, by Olympus (1 mentions)
Syto green, by Thermo Fisher Scientific (1 mentions)
2 protocols using fluoview software
1
Immunocytochemical Staining of Lysosomes
2
Quantitative Analysis of NET Formation
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Qualitative assessment of NET formation was performed, as previously referenced (10 (link), 17 (link)). Briefly, participant primary PMNs were incubated with control buffer or stimulated with LPS (100 ng/mL) for 2 h at 37°C in 5% CO2/95% air on glass coverslips coated with poly-l -lysine. After stimulation, PMNs were gently washed with PBS and incubated with a mixture of cell permeable (Syto Green, Molecular Probes) and impermeable (Sytox Orange, Molecular Probes) DNA fluorescent dyes. Confocal microscopy was accomplished using a FV300 1X81 Microscope and the FluoView software (Olympus). Both 20× and 60× objectives were used. Z-series images were obtained at a step size of 1 μm over a range of 20 μm for each field. The Olympus FluoView software and the Adobe Photoshop CS2 software were used for image processing. Semiquantitative analysis of NET formation was accomplished using the ImageJ analysis software (NIH) and a standardized grid system with rigorous NET quantitation. Statistical comparisons were made via one way ANOVA with Tukey’s multiple comparisons post hoc testing.
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