Goat anti rabbit igg coupled to alexa fluor 546

Rabbit anti-human GCDH antibody was kindly provided by Dr. S. I. Goodman (University of Colorado Health Sciences Center, Denver). The polyclonal mouse anti-human DLST and rabbit anti-human ETFA antibodies were purchased from Sigma (Munich, Germany), rabbit anti-human ETFB from Abcam (Cambridge, UK), and rabbit anti-LC3 from Abgent (San Diego, USA). The monoclonal mouse anti-GFP antibody was obtained from Roche (Mannheim, Germany) and rabbit anti-MnSOD from Millipore (Billerica, USA). Peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG was from Dianova (Hamburg, Germany). HRP-conjugated anti-V5 antibody, monkey anti-mouse IgG coupled to Alexa Fluor 488 and goat anti-rabbit IgG coupled to Alexa Fluor 546 were from Invitrogen (Karlsruhe, Germany).

To visualize phagocytosis under conditions of proinflammatory LPS and PAL stimulation and its modulation by 5-AZA and SAM methylation modulators, we used confocal microscopy. SIM-A9 cells were seeded on sterile coverslips in six-well plates covered with 0.01% poly-L-lysine solution (Sigma, p4707). After treatment, cells were fixed by adding 4% paraformaldehyde (PAF) (Sigma, 158127) for 10 min, washed three times with 1 × PBS, and were permeabilized with PBS solution 1 × + 0.1% Triton X-100 (PBST) (Sigma, × 100) for 10 min. Next, the cells were blocked in PBST + 10% goat serum (GIBCO, 16210064) for 30 min and washed three times with 1 × PBS. Microglia immunodetection was performed by overnight primary antibody anti-mouse Iba-1 (1:200) (Abcam, ab178847) incubation at 4°C followed by goat anti-rabbit IgG coupled to Alexa Fluor 546 (1:1000) (Invitrogen, A-11035). Finally, cells were washed 3 × with 1 × PBS and mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories, H-1000-10). Fluorescent signals were detected by confocal-laser microscopy using an Olympus BX61W1 microscope with an FV1000 module with diode laser. Finally, the images were processed with ImageJ software.