Immunofluorescence Staining Protocol

Sections were stained for the following antibodies: Aquaporin 4 (AQP4), Glial Fibrillary Acidic Protein (GFAP), ionized calcium-binding adaptor molecule 1 (IBA1), or NLR family pyrin domain containing 3 (NLRP3) (Table 1). More specifically, free-floating sections were washed three times for 5 min each in 1X PBS and then permeabilized in PBS with 0.3% Triton X (PBX) for 30 min at room temperature. Samples were blocked in either 2% bovine serum albumin (AQP4, GFAP, or IBA-1) or 5% Normal Donkey Serum (NLRP3) in PBS for 1 h at room temperature. Once samples were permeabilized and blocked, they were incubated for 16–18 h at 4°C with a primary antibody. The following day, sections were washed three times for 5 min in PBX and incubated for 1.5 h at room temperature with secondary antibodies Alexa Flour 488 Goat anti-mouse IgG antibody (Invitrogen, Carlsbad, California) (GFAP) or Alexa Flour 546 Goat anti-rabbit IgG antibody (Invitrogen, Carlsbad, California) (AQP4, IBA-1, and NLRP3). After three more 5-min PBX washes, samples were mounted and coverslipped with Slow Fade Reagent with DAPI (Invitrogen, Carlsbad, CA). Sections were then imaged using a Zeiss fluorescence microscope at 20X magnification by an investigator blinded to animal groups.