Immunofluorescence Staining of THP-1 Macrophages

For immunofluorescence experiments [23 (link),26 (link)], cells were processed as previously described, with slight modifications [23 (link),26 (link)]. Briefly, THP-1 macrophages were incubated for 1 h and immediately fixed with pre-warmed 3.7% formaldehyde (15 min, room temperature). After permeabilization (0.2% Triton-X-100, 10 min), unspecific binding sites were blocked (2% Donkey Serum, 0.5% Bovine Serum Albumine, 1 h). Afterwards, cells were incubated for 2 h with anti-Caveolin-1 antibody (Abcam, ab192452, 1:500), anti-TLR4 antibody [76B357.1] (Abcam, ab22048, 1:200) and anti MIF antibody (Abcam, ab7207, 1:500). After 5 washing steps, species-specific fluorescence-labeled antibodies were used for localization (Donkey Anti-Goat IgG (H+L), Alexa Fluor^® 647 (705-605-003) and Donkey Anti-Mouse IgG (H+L) Alexa Fluor^® 488 (715-545-150) from Jackson ImmunoResearch Laboratories, Inc., Pennsylvania, US and Donkey anti-Rabbit IgG (H+L) polyclonal secondary antibody, Alexa Fluor^® 568 (A10042) from Thermo Fisher Scientific, Waltham, US).