Alexa fluor 488 conjugated goat anti mouse igg secondary antibody

For the surface marker staining, cells were stained according to the manufacturer's directions. The following antibodies were used: anti-F4/80 (eBioscience), anti-CD80 (eBioscience, Waltham, MA, USA), anti-CD86 (eBioscience), anti-MHCII (eBioscience). To detect LC3II in splenic macrophages, Flowcellect autophagy LC3 antibody-based assay kit (Merk Millipore) was used according to the manufacturer's directions. To assess TLR7, Notch1 expression in macrophages, spleen cells were then stained with anti-F4/80 and resuspended with fixation/permeabilization solution (eBioscience) then stained with anti-TLR7 (IMGENEX, CO, USA), anti-Noch1 (eBioscience), respectively. Similarly, fixed and permeabilized cells were stained with anti-Hes-1 (abcam) or anti-P62 (Merk Millipore), and then stained with Alexa Fluor 647 conjugated goat anti-rabbit IgG secondary antibody (abcam) and Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody (abcam), respectively. Annexin V-FITC and PI Apoptosis kit (eBiosciences) was used to examin the mortality of macrophages in vivo and in vitro. All of the flow cytometry data were aquired with the BD FACS Calibur cytometer and analyzed by FlowJo software.

The control and H₂O₂ treated CEFs, EGK.X blastoderm cells, and PGCs were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After blocking the non-specific binding with 1% bovine serum albumin (Sigma-Aldrich) for 1 h, cells were incubated with mouse anti-γ-H2A.X antibody (phospho S139) (Abcam, Cambridge, UK) at a 1:200 dilution in blocking buffer for overnight at 4 °C to detect the cells undergoing DNA repair. Cells were then incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Abcam) at a 1:500 dilution in the blocking buffer for 1 h. Cells were counterstained with DAPI and analyzed under a fluorescence microscope (Eclipse Ti).