Dylight 488 labeled secondary antibody

For immunostaining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS, then the cells were incubated with primary antibodies(anti-NLRP3 antibody (1:100), anti-KAT5 antibody (1:100), anti-GOLGA4(1:200) or anti-TGN38(1:200)) followed by staining with secondary antibody(DyLight 488-labeled secondary antibody (Invitrogen)(1:50),Alexa Fluor 594-conjugated secondary Ab (Invitrogen) (1:50),Alexa Fluor® 488 Goat anti-mouse IgG (minimal x-reactivity) Antibody(1:100) or Cy3–conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:100)), while nuclei were stained with DAPI containing mounting medium (Beyotime). To better preserve the dTGN structures in COS-7 cells, 0.01% Triton X-100 and 0.1% saponin were respectively in place of 0.1% Triton X-100 in permeabilization step for immunostaining of GOLGA4 and TGN38. Fluorescence images for fixed cells were taken with confocal fluorescence microscope (SpinSR10; Olympus) and (LSM800; ZEISS).

Primary macrophages were stimulated with LPS for indicated hours, then cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 3% BSA for 1 h, cells were incubated overnight with anti-NLRP3 antibody (1:100 in PBS containing 3% BSA) and β-TrCP1 antibody (1:100 in PBS containing 3% BSA), followed by staining with DyLight 488-labeled secondary antibody (Invitrogen) and Alexa Fluor 594-conjugated secondary Ab (Invitrogen) (1:50 in PBS containing 3% BSA). Nuclei were co-stained with DAPI (Invitrogen). Stained cells were viewed using a confocal fluorescence microscope (SpinSR10; Olympus).