Fitc conjugated goat anti rabbit igg antibody

A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). To induce anoxia, cells were placed in an anoxic chamber with 5% CO₂ and 95% N₂ at 37 °C for 2 h followed by reoxygenation for 2 h. Exogenous rhMG53 was conjugated with Rhodamine by a dye labeling kit (GBiosciences, St Louis, US.), and applied to cells immediately after anoxia. After the reoxygenation period, the cells were used for Annexin V staining or immunofluorescence staining. Cells were fixed with 4% paraformaldehyde (30 min), and custom rabbit anti-MG53 antibody was applied (1:200 dilution) over night at 4 °C, which was followed by FITC–conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratory, West Grove, Pa). Immunofluorescence confocal images were acquired (Olympus AX70 laser confocal microscopy) at excitation wavelengths of 350 nm and 507 nm; emission was detected at 450 and 529 nm. Cells that were treated with only FITC–conjugated goat anti-rabbit IgG antibody revealed no immunofluorescence, and omission of the anti-MG53 antibody showed no green fluorescence after merging the images.

Annexin A5 content in mouse-derived CDNPs was evaluated by Western blot. 15µl isolated CDNPs were mixed with 5µl 4× Laemmli buffer and heated at 60°C for 20 min. Samples were then run on a 10% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific, Carlsbad, CA) in MES buffer (Bio-Rad, Hercules, CA). After transferring the protein to a PVDF membrane for 60 min at 75 mA, the membrane was blocked with 3% BSA-solution for 60 min at room temperature. The first, second and third antibodies were diluted in 3% BSA as well. The primary Anti-Annexin A5 antibody (GeneTex, Irvine, CA) was applied in a 1:300 dilution and incubated overnight at 4°C. The membrane was washed with Tween-PBS and incubated with the second FITC-conjugated goat anti rabbit IgG antibody (1:200; Jackson Immuno Research, West Grove, PA) for 90 minutes at room temperature. After a washing step, the membrane was incubated for 60 min with the tertiary alkaline phosphatase-conjugated mouse anti-FITC antibody (1:5000; Jackson Immuno Research, West Grove, PA) at room temperature. Finally, washed membranes were developed using 1 ml ECF substrate (GE Healthcare, Little Chalfont, United Kingdom).