Cy5 conjugated goat anti rat igg

BMMs were fixed by 4% paraformaldehyde and penetrated by 0.2% Triton X-100 (Amresco, OH, USA). After being blocked with 2% BSA (Roche, Switzerland), they were incubated with rabbit anti-ASC polyclonal antibody (AG-25B-0006, 1:400, Adipogen, San Diego, USA). FITC-conjugate affinipure goat-anti-rabbit IgG (111-095-144, 1:400, Jackson Immunoresearch, PA, USA) was used as a secondary antibody. Nuclei were stained with DAPI.
The liver specimen was fixed in 4% paraformaldehyde, and frozen sections of 5 μm were used for immunofluorescence. Frozen sections were incubated with rat anti-F4/80 polyclonal antibody (sc-71085, 1:400, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-NLRP3/NALP3 monoclonal antibody (AG-20B-0014, 1:400, Adipogen, San Diego, USA) as the first antibody. Cy5-conjugated goat anti-rat IgG (1:400, 112-175-143) and Cy3-conjugated goat anti-mouse IgG (1:400, 115-165-062) was from Jackson ImmunoResearch Laboratories and used as a secondary antibody. Finally, the sections were stained with DAPI and observed under a confocal microscope (LSM510, Carl Zeiss MicroImaging, Jena, Germany).

Immunofluorescence was performed as previously described^{19 (link)}. Briefly, immunofluorescence detection was conducted with antibodies against RCP (1:500, Cell Signaling Technology), E-cadherin (1:500, Santa Cruz Biotechnology), β1 integrin (1:500, Santa Cruz Biotechnology), MT1-MMP (1:500, Santa Cruz Biotechnology), and LAMP1 (1:500, Abcam, Cambridge, UK) overnight. The cells were washed with ice-cold PBS and incubated with Cy3-conjugated goat anti-rabbit IgG (1:500, Jackson ImmunoResearch, PA), Cy2-conjugated goat anti-mouse IgG (1:500, Jackson ImmunoResearch) and Cy5-conjugated goat anti-rat IgG (1:500, Jackson ImmunoResearch). The nuclei of cells were marked with 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen). The cells were observed by confocal microscopy (x600, LSM710, Carl Zeiss).

Liver, lung, and kidney sections were fixed, stained, and imaged using confocal microscopy as previously described (18 (link), 31 (link)). Tissues were incubated with specific primary antibodies as follows: Ly6G (2 μg /ml; BD Bioscience; cat no. 560599), citrullinated histone H3 (5 μg/ml, rabbit IgG; Abcam ab5103), CD41 (5 μg/ml, rat IgG; Abcam, ab33661), fibrinogen (2 μg/ml, sheep IgG; Abcam ab61352). Sections were then incubated with Alexa 488-conjugated F-actin phalloidin (1:500, Invitrogen, San Diego, CA, USA) in the presence of the following secondary antibodies depending on the primary antibody pairing: Cy5–conjugated goat anti-rat IgG (1:1000, for anti-CD41 antibody, Jackson Immunoresearch 112-165-167); 488-conjugated goat anti-rat IgG (1:500 for CD41 antibody, Molecular Probes, A11006) for 1 h. A Hoeschst nuclear stain was applied for 30 s and slides were prepared for imaging. Imaging conditions were maintained at identical settings within each antibody-labeling experiment with original gating performed using the negative control. Large area images in X and Y using a Nikon A1 confocal microscope (purchased with 1S10OD019973-01 awarded to Dr. Simon C. Watkins). Quantification was performed using NIS Elements citH3 staining co-localized with Ly6G staining and normalized by area of actin.