Rabbit asc

Twenty μm thick coronal sections were washed with PBS and 0.4% Triton-X PBS for 20 minutes. The sections were then blocked with 10% normal donkey serum for 1 hour at room temperature in PBS containing 0.1% Triton X-100, followed by incubation with primary antibody for 1–3 nights at 4°C in the same buffer. Primary antibodies used for this study included rabbit-NLRP3 (Santa-Cruz Biotechnology, sc-66846), rabbit-ASC (Santa-Cruz Biotechnology, sc-22514-R), goat-cleaved caspase-1 (Santa-Cruz Biotechnology, sc-22165), rabbit-IL-1β (Abcam, ab9722), goat-Iba1 (Abcam, ab5076), rabbit-P2X7 receptor (Sigma, P8232), and mouse-NeuN (Millipore, MAB377). After primary antibody incubation, sections were washed for 3 × 10 minutes at room temperature (RT), followed by incubation with the appropriate secondary antibody: Alexa-Fluor488/568/647 donkey anti-rabbit/anti-mouse/anti-goat (Invitrogen) RT/1 hour. Sections were then washed with PBS containing 0.1% Triton X-100 for 3 × 10 min, followed by 2 × 5 min with 1x PBS and briefly with water. The sections were then mounted with water-based mounting medium containing antifading agents and observed using confocal microscopy. All images were captured on a confocal laser microscope (Carl Zeiss, Germany) using the Zen software at 40x magnification and 50 μm scale bar.

The proximity ligation (Duolink) assay was performed, as described by our laboratory [44 (link)]. Briefly, coronal brain sections were blocked in 5% (vol/vol) donkey serum for 1 hour at room temperature and incubated overnight with the following pairs of primary antibodies: goat-NLRP3 (Santa Cruz, sc-34,408) and rabbit-ASC (Santa Cruz, sc-22,514-R); or rabbit-NLRP3 (Santa Cruz, sc-66,846) and goat-TXNIP (Santa Cruz, sc-33,099) at 4°C. These sections were then incubated for 1 hour at 37°C with the following Duolink PLA probes: anti-Rabbit MINUS (Sigma-Aldrich, DUO92005) and anti-goat PLUS (Sigma-Aldrich, DUO92003). Duolink in situ detection reagent kit (Sigma-Aldrich, DUO92008) was used for ligation and amplification at 37°C using the according to the manufacturer's protocol. All sections were then mounted on a slide using DAPI-mounting media, and all images were captured on a confocal laser microscope (Carl Zeiss, Germany) using the Zen software at 40x magnification. Fluorescence of PLA indicating interacting proteins was analyzed as intensity above threshold using NIH ImageJ software and represented as fold change from shams.

Tissue sections were blocked in 5% (vol/vol) donkey serum for 1 hour at room temperature and incubated overnight with primary antibodies, goat-NLRP3 (Santa-Cruz Biotechnology, sc-34408), and rabbit-ASC (Santa-Cruz Biotechnology, sc-22514-R) at 4°C. These sections were then incubated with Duolink PLA probes, anti-Rabbit MINUS (Sigma-Aldrich, DUO92005) and anti-goat PLUS (Sigma-Aldrich, DUO92003) for 1 h at 37°C. Ligation and amplification were carried out at 37°C using the Duolink in situ detection reagent kit (Sigma-Aldrich, DUO92008) according to the manufacturer's protocol. All sections were then mounted on a slide and all images were captured on a confocal laser microscope (Carl Zeiss, Germany) using the Zen software at 40x magnification and 50 μm scale bar.