Mouse anti asc

Immunofluorescent staining was performed as previously described [25 (link)]. Briefly, tissue sections or cells were blocked using a 10% bovine serum albumin solution with 0.25% triton X-100 for 1 h at room temperature, followed by incubation overnight at 4 °C with primary antibodies. These primary antibodies included rabbit anti-NeuN (1:500, Millipore), rabbit anti-GFAP (1:500, Cell Signaling Technology), rabbit anti-IBA-1 (1:500, Wako Pure Chemical Industries), goat anti-IBA-1 (1:500, Wako Pure Chemical Industries), mouse anti-GSDMD (1:50, Santa-Cruz), mouse anti-ASC (1:200, Santa-Cruz), mouse anti-caspase-1 (1:200, Santa-Cruz), mouse anti-NLRP3 (1:200, AdipoGen), rabbit anti-Hv1 (1:50, Sigma), and mouse anti-8-OHdG (1:200, Abcam). Next, samples were washed in PBS and incubated at room temperature for 1 h with the appropriate secondary antibodies, as follows: FITC-conjugated goat anti-mouse immunoglobulin G (IgG), Cy3-conjugated goat anti-rabbit IgG, Cy3-conjugated rabbit anti goat IgG, Cy3-conjugated rabbit anti-rat IgG, and 488-conjugated donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA). Finally, sections were stained with 4,6-diamidino-2-phenylindole (DAPI). Samples were imaged using a confocal microscope (Olympus, BX51).

LX-2 cells were infected with B. abortus, and after 24 h, cells were fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.3% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany) for 10 min, and blocked with PBS containing 1% BSA for 1 h. Infected cells were stained with mouse anti-ASC (Santa Cruz Biotechnology) diluted in 0.1% PBS–Tween 20 overnight at 4°C. Cells then were incubated with rabbit anti-mouse Alexa Fluor 488 (Molecular Probes, Life Technologies) diluted in 0.1% PBS–Tween for 4 h at room temperature. 4,6-Diamidine-2-phenylindole (DAPI) was used for nuclear staining, and cells were stained for 30 min at room temperature. After washing in PBS, cells were mounted and then analyzed by fluorescence microscopy. Confocal images were analyzed using FV-1000 confocal microscope with an oil immersion Plan Apochromatic 60 × NA1.42 objective (Olympus).

We visualized NLRP3 inflammasomes in microglia, neurons, and astrocytes through triple immune-fluorescence staining of brain tissue sections (n=6/group) for NLRP3, an apoptosis-associated speck-like protein containing a CARD (ASC), and IBA-1, NeuN, or GFAP. Goat anti-NLRP3 (1:500, Millipore), mouse anti-ASC (1:1000, Santa Cruz), rabbit anti-NeuN (1:1000, Millipore), rabbit anti-GFAP (1:1000, Millipore), rabbit anti-iba1 (1:1000 Abcam), donkey anti-goat IgG tagged Alexa Fluor 488 (1:200, Invitrogen), donkey anti-mouse Alexa Fluor 594 (1:200, Invitrogen), and donkey anti-rabbit 405 (1:200, Invitrogen) were employed in these studies. Two-micrometer thick optical Z-sections were employed for blinded quantification of the total number of NLRP3 inflammasomes (i.e., structures positive for both NLRP3 and ASC) in 216 μm² area of the CA3 subfield of the hippocampus (2 sections/animal, n=5-6/group). We also quantified the percentages of IBA-1+ microglia, NeuN+ neurons, and GFAP+ astrocytes containing NLRP3 inflammasomes (2 sections/animal) [25 (link)].

Cell lysates and methanol-chloroform precipitated cell-free supernatants were separated using 4-20% Mini-Protean TGX Precast Protein Gels (Bio-Rad, Hercules, CA, USA), and transferred onto PVDF membranes (Bio-Rad). Membranes were blocked with fish gelatine (Norland products, Cranbury, NJ, USA) and then blotted with goat anti-IL-1β (R&D Systems AF-201-NA, 1:1000), rabbit anti-C4BP (Agrisera custom made, 1:5000), rabbit anti-tubulin (Abcam, Cambridge, UK; ab6046, 1:5000) or mouse anti-ASC (Santa Cruz, Dallas, TX, USA; B-3, 1:1000). Immunoblots were developed with appropriate horse radish peroxidase (HRP)-conjugated secondary antibodies (DAKO, Glostrup, Denmark; 1:5000), and subsequently developed using Immobilon ECL (Millipore, Darmstadt, Germany). Immunoblot targets from the same sample were detected on the same membrane, which was either cut and targets immunoblotted in parallel, or stripped and re-probed. Stripping of HRP activity was achieved with 30% H₂O₂ for 15 minutes at room temperature, prior to re-incubation with a subsequent primary antibody.

The interactions of endogenous ASC, ZBTB16, SUMO1 and NLRP3 were assessed in macrophage cells by proximity ligation assay (PLA, Duolink) according to the manufacturer’s instructions (Sigma-Aldrich). BMDMs were fixed with ^-20 °C methyl alcohol and then permeabilised with 0.3% Triton X-100 before the addition of the specified antibody pairs mouse anti-ASC (Santa Cruz) with rabbit anti-ZBTB16 (Bioss) or rabbit anti-SUMO1 (Cell Signalling Technology) or, alternatively, rabbit anti-ASC (AdipoGen) with mouse anti-NLRP3 (AdipoGen). The images were captured by Zeiss LSM 880 confocal fluorescence microscope at 63x magnification with Airyscan image processing and analyses with ImageJ software.

For NLRP3/ASC double immunofluorescence, rabbit anti-NLRP3 (dilution 1:100, ABclonal Technology, Wuhan, China) and mouse anti-ASC (dilution 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, United States) primary antibodies were used. Sections were then labeled with FITC– and Cy3–conjugated secondary antibodies (dilution 1:200, Servicebio, Wuhan, China) for 2 h at room temperature, avoiding light, followed by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) staining solution (Servicebio, Wuhan, China). Subsequently, the anti-fluorescence quencher was used to seal the section (Servicebio, Wuhan, China). Fluorescence images were photographed with a Zeiss LSM 880 confocal microscope (Carl Zeiss, Jena, Germany).