Mca711g

PFA-fixed brain sections were washed twice with DPBS (without calcium and magnesium) before undergoing fixation with 95% ethanol (pre-chilled to 4 °C) for 10 min. Antigen retrieval was performed with a 15-min incubation of pre-warmed (37 °C) trypsin (0.25%) before blocking endogenous peroxidase activity with a 5-min incubation of 0.3% hydrogen peroxide and 0.3% normal rabbit serum in DPBS. Sections were blocked in DPBS with 1.5% normal rabbit serum for 20 min before primary antibody incubation with 1:100 rat anti-mouse CD11b (Bio-Rad MCA711G, Hercules, CA, USA) in DPBS with 2.5% normal rabbit serum. The primary antibody was visualized with the avidin-biotin horseradish peroxidase technique in accordance with the manufacturer’s instructions (Vector Laboratories, Vectastain Elite Kit PK6104, NovaRED Peroxidase (HRP) Substrate Kit SK-4800, Burlingame, CA, USA). Sections were then counterstained with hematoxylin in accordance with the manufacturer’s instructions (Vector Laboratories, H-3401, Burlingame, CA, USA), and mounted with Vectamount Permanent Mounting Medium (Vector Laboratories, H-5000, Burlingame, CA, USA). Brightfield images were subsequently obtained using a Zeiss Axio Scan.Z1 slide scanner with the ZenBlue software (Carl Zeiss, Jena, Germany).

Mice were anesthetized with an intraperitoneal (i.p.) injection of Avertin (300 mg/kg) and transcardially perfused with PBS followed by 4 % paraformaldehyde. Brains were dissected and fixed in 4% paraformaldehyde overnight, cryoprotected in 15% (w/v) then 30% sucrose, and frozen in OCT compound (Tissue-Tek). Serial 30 μm coronal sections were generated using a cryostat. Free-floating sections were washed, blocked (0.2% Triton-X-100 + 5% normal goat serum (NGS) in PBS) and stained with anti-Cd11b (1:50, rat, MCA711G, Bio-Rad) and anti-rabbit GFAP (1:500; rabbit, #7260, Abcam) antibodies diluted in PBS containing 0.1 Triton X-100 + 1% NGS overnight at 4 °C. Sections were then washed and subsequently stained with secondary antibodies (goat anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 546, 1:500, ThermoFisher Scientific). Sections were mounted with SlowFade Diamond mountant containing DAPI (Life Technologies) and imaged with a 10× objective on a Zeiss LSM710-Duo confocal microscope.

Animals were perfused with 4% PFA and brains removed for histology. Rapidly frozen 30 µM brain sections were incubated with primary CD11b antibody (Bio-Rad, MCA711G) or GFAP, NLRP3 overnight at 4 °C. Secondary Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, A-11008) or Alexa Fluor 594 goat anti-mouse (Invitrogen, A-11032) was added for 2 h, followed by the mounting of sections with DAPI (Invitrogen, 3693). Fluorescent images were acquired at room temperature on a Zeiss Observer. A Z1 inverted microscope (Carl Zeiss, Oberkochen, Germany) was used; images were processed using AxioVs 40 4.8.0.0 software (Carl Zeiss MicroImaging, Oberkochen, Germany). Photographs were acquired using an AxioCam MRm digital camera (Carl Zeiss, Oberkochen, Germany).