A0237

Immunofluorescence was performed as previously described.⁷ The rats’ brains were fixed with 4% paraformaldehyde and then cut into 20‐mm sections. The following primary antibodies were used: anti‐NLRP3, 1:50, ab4207, Abcam; anti‐p65, 1:50, 10745‐1‐AP, Proteintech; anti‐Iba‐1, 1:200, ab5076, Abcam; anti‐GFAP, 1:50, A0237, AbClonal. Fluorescence‐labeled secondary antibodies were applied and 4′,6‐diamidino‐2‐phenylindole (DAPI, Invitrogen) was used to stain the nuclei. Samples were visualized using a TCS SP5 multiphoton laser scanning confocal microscope (Nikon, Tokyo, Japan).

Isolated MGCs were plated on gelatin-coated imaging plates (Eppendorf; Enfield, CT) and grown to confluency. Cells were washed with DPBS, fixed with ice-cold 80% acetone for 10 min at RT, and permeabilized with 0.25% Triton X-100 for 10 min at RT prior to blocking in 10% normal goat serum (Life Technologies) for 2–4 h at RT. Cells were then incubated with primary antibodies at 4 °C overnight: anti-IL6Rα antibody (sc-373708, Santa Cruz, 1:100), anti-vimentin antibody (5741S, Cell Signaling, 1:100), or anti-GFAP antibody (A0237, ABclonal, 1:100). For double labeling of IL-6Rα and vimentin, MGCs were incubated with both primary antibodies simultaneously. Cells were washed three times with DPBS and then incubated with respective secondary antibodies conjugated to Alexa Fluor 488 (Donkey anti-Rabbit, A21206, Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 546 (Donkey anti-Mouse, A10036, Invitrogen) for 1 h at RT. Cells were washed three times and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min (1:2000, Thermo Scientific). Images were acquired using a Leica STELLARIS Confocal Microscope (Buffalo Grove, IL, USA) at 40× magnification.