Cf568 conjugated goat anti rabbit igg

Immunofluorescence analysis was performed as previously described [16] (link). Cells were fixed in 4% paraformaldehyde, permeabilized with ice-cold 100% methanol, and immunostained with mouse anti-β-actin and rabbit anti-p65 antibodies followed by CF488-conjugated goat-anti-mouse IgG and CF568-conjugated goat-anti-rabbit IgG, highly cross-absorbed (Biotium, Hayward, CA). Nuclei were revealed by Hoechst 33342 staining. Fluorescence images were collected under a Leica DMIRB fluorescent microscope (Leica Microsystems, Wetzlar, Germany) armed with a Spinning Disk Confocal Microscopy system (UltraView cooled CCD; Perkin Elmer, Waltham, MA, USA).

Immunofluorescence was performed essentially as previously reported [27 (link), 28 (link)]. Briefly, cells were incubated with appropriate primary antibodies followed by incubation with CF488-conjugated goat-anti-mouse IgG and CF568-conjugated goat-anti-rabbit IgG (Biotium, Hayward, CA, USA). Fluorescence images were collected under a fluorescent microscope armed with a Spinning Disk Confocal Microscopy system (Ultra View cooled CCD; Perkin Elmer, Waltham, MA, USA).

Bone marrow-derived macrophages were planted in glass-bottomed dishes (5 × 10⁵ cells/dish) and cultured at 37°C overnight. Then the cells were primed with 500 ng/ml LPS for 4 h, followed by treatment with 2 mM metformin or vehicle (PBS) for 1 h. Subsequently, 2 mM ATP was added to the culture medium for 30 min. After these treatments, the cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 2 ml cold methanol (−20°C) for 10 min. Then the cells were incubated with ASC antibody (1:300) overnight, followed by staining with CF568-conjugated goat-anti-rabbit IgG (#20103) (Biotium, Hayward, CA, USA) for 1 h. Finally, Hoechst 33342 solution (5 µg/ml in PBS) was added to stain the nuclei for 10 min. The cells were observed under a Zeiss Axio Observer D1 microscope with a Zeiss LD Plan-Neofluar 40×/0.6 Korr M27 objective (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Fluorescence images were captured with a Zeiss AxioCam MR R3 cooled CCD camera controlled with ZEN software (ZEISS).