Alexa fluor488 568 647 donkey anti rabbit anti mouse anti goat

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.

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 2 nights at 4°C in the same buffer. Primary antibodies used for this study included mouse monoclonal CD68 (Abcam, ab31630, 1 : 500), goat polyclonal CD206 (Santa Cruz Biotechnology, sc-34577, 1 : 50), rabbit monoclonal iNOS (Cell signaling, D6B6S, 1 : 400), rabbit monoclonal IL1RA (Abcam, ab124962, 1 : 400), and goat polyclonal Iba1 (Abcam, ab5076, 1 : 400). After primary antibody incubation, sections were washed for 3 × 10 minutes at room temperature, followed by incubation with the appropriate secondary antibody: Alexa-Fluor488/568/647 donkey anti-rabbit/anti-mouse/anti-goat (1 : 200) (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 distilled H₂O. 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 magnifications and 50 μm scale bar.