protocol – ddewipputri

Cover glasses were prepared on a 24-well plate and were treated with Poly-L-lysine solution (Sigma) for seeding cells. The cells were stimulated with ligand cGAS-STING (ISD (1μg/ml)-lipofectamine 2000), or several TLR ligand, for various time points. Then, cells were washed with PBS and fixed with 4% paraformaldehyde (Nacalai Tesque) for 20 minutes at room temperature. After washing three times with 0.02% Triton X-100 in PBS, 100 mM glycine in 0.02% Triton X-100/PBS was added at room temperature for 30 minutes. Cells were washed 3 times with 0.02% Triton X-100 in PBS and were blocked with 10% FBS, 0.02% Triton X-100/PBS for 1h at room temperature. Cells were incubated with primary antibodies diluted with 10% FBS 0.02% Triton X-100 at 4 °C overnight. After washing three times with 0.02% Triton X-100, cells were applied with the secondary antibodies diluted with 10% FBS in 0.02% Triton X-100 and were incubated for 1 hour. After three times washing with 0.02% Triton X-100, Hoechst 33342 (Dojin) was diluted with 10% FBS in 0.02% Triton X-100 and incubated for 10 minutes. After 3 times washing with 0.02% in Triton X-100, samples were enclosed on slide glasses with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type (Nacalai Tesque). The prepared samples were observed with confocal fluorescence microscope LSM 700 (ZEISS), and images were processed with ZEN software. Dilution ratios of the primary and secondary antibodies are shown below.