Immunofluorescence assay was used to detect the number of γ-H2AX foci. Cells were seeded in 24-well plates. After washing with PBS, cells were fixed in 3% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS. The cells were then stained with γ-H2AX primary antibody (Cell Signaling Technology; 1:200) and thereafter the secondary antibody (fluorescein goat anti-rabbit IgG, Invitrogen; 1:1000). Cells were stained with a DAPI (Antifade Mounting Medium with DAPI, Beyotime). The images were captured using an Olympus BX60 fluorescent microscope (Olympus America Inc).
Fluorescein goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Fluorescein goat anti-rabbit IgG is a secondary antibody conjugated with the fluorescent dye fluorescein. It is designed to detect and bind to rabbit immunoglobulin G (IgG) molecules in various immunoassay applications.
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2 protocols using fluorescein goat anti rabbit igg
1
Immunofluorescence Assay for γ-H2AX Foci
2
Immunofluorescence Analysis of P2X7R and Arg-1 in G- and M-MDSCs
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G- and M-MDSCs were centrifuged on polylisine slides, dried at RT and fixed in 2% paraformaldehyde in PBS (pH 7.4) for 20 min at RT. After washing with PBS, the slides were incubated with ammonium chloride 100 mM for 20 min at 4 C°, permeabilized with Triton X-100 (0.1% in PBS) for 8 min at RT and blocked with BSA (2% in PBS) for 20 min at RT. Cells were then incubated for 1 h with anti-P2X7R and anti-ARG-1 Abs (Sigma Aldrich and Santa Cruz Biotechnology, respectively). Binding of primary antibodies was detected with fluorescein goat anti-rabbit IgG (Invitrogen). After rinsing, the slides were counterstained with DAPI (Vectashield mounting, Vector Laboratories, Orton Southgate, Peterborough, UK) and coverslipped. Digital images were acquired using a Nikon E-1000 fluorescence microscope (Nikon Instruments, Tokyo, Japan) equipped with appropriate filter sets and the Genikon imaging system software (Nikon Instruments).
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