Goat nlrp3

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.