Structured illumination

Astrocytes isolated from C57B6J mice were cultured on glass coverslips and treated with IL-1β (100 ng/ml; Millipore), IL-1β + altenusin (50 µM; Enzo Life Sciences), IL-1β + GW4869 (20 µM; Calbiochem), IL-1β + IL-1 receptor agonist (100 ng/ml), TNFα (100 ng/ml), or IL-10 (100 ng/ml) for 2 to 60 min. Cells were fixed with 4% PFA, and lipid raft membrane microdomains were identified using a cholera toxin subunit B conjugated to Alexa Fluor 555, which binds the ganglioside GM1 (CTB-555, Invitrogen/Molecular Probes) (30 (link), 59 (link)). CTB-555–labeled cells were incubated with primary antibodies to ceramide (1:200; Santa Cruz Biotechnology) or nSMase (1:200; Santa Cruz Biotechnology), and the corresponding secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 546 (1:1000; Invitrogen/Molecular Probes). Fluorescence was imaged with a 100× objective by optical sectioning using structured illumination (Carl Zeiss Inc.). All images for quantification were taken with identical settings and performed on a single plane of focus through the brightest point. Colocalization was confirmed by three-dimensional reconfiguration of z-stack images using orthogonal views as previously described (31 (link)).

EVs released from astrocytes of GFAP-EGFP mice were visualized by fluorescence imaging of EGFP⁺ puncta in liver, spleen, and lung tissue sections. Tissues were postfixed in 4% PFA and cryoprotected in 30% sucrose, and 20-µm sections were cut using a cryostat microtome (Leica). Nonspecific binding was blocked with 5% normal goat serum plus 5% normal horse serum in tris-buffered saline (TBS) containing 0.1% Triton X-100 (Fisher Scientific). EGFP was enhanced by incubating sections with anti-GFP rabbit sera (1:500; Invitrogen) at 4°C overnight, followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 (1:1000; Invitrogen). Cells were visualized with F-actin (1:100; Abcam), and nuclei were stained with DAPI as previously described (60 (link)). Imaging was performed with 40× and 100× objectives using optical sectioning by structured illumination (Carl Zeiss Inc.).