Slowfade reagent with dapi

To localize EST6 in the antenna, we performed immunohistochemistry with the anti-EST6 antibody above on transgenic flies expressing RFP under the control of either the Orco or lush promoter or GFP under the control of the elav promoter. Est6 null mutant flies were used as a control for the specific labelling of the antibody. Specifically, heads with antennae still attached from 5-day-old Orco^Gal4/UAS-mCD8::RFP, elav^LexA/LexAOP-mCD8::GFP, lush^Gal4/UAS-mCD8::RFP or Est6 null mutant males were fixed for 3 h in 4% paraformaldehyde with 0.2% Triton X-100, then washed for 1 h with PBS containing 0.2% Triton X-100 (PBST). The heads were then embedded in Tissue-Tek^TM (CellPath) and cryosections (15 μm) were set in cell culture insert (Greiner Bio-one). After blocking with 3% normal goat serum and 1% BSA in PBST (1 h at room temperature), the anti-EST6 antibody was diluted from 1:3,000 to 1:750 (v:v) in the blocking solution (3% normal goat serum in PBST) and incubated overnight at room temperature. After a brief rinse in PBST, an anti-mouse conjugated Alexa-488 or Alexa-596 (Invitrogen) was applied at a concentration of 1:800 (v:v) in the blocking solution for 4 h at room temperature. Tissues were mounted in Slowfade reagent with DAPI (Invitrogen). Images were captured on a Leica SP5 confocal microscope and analysed using ImageJ 1.47 v (http://imagej.nih.gov/ij).

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.