Alexa fluor 488 or 594 labeled secondary antibodies

For western blot and histological analyses, monoclonal mouse antibodies against α-SG (NCL-a-SARC), β-SG (NCL-b-SARC), and γ-SG (NCL-g-SARC) were purchased from Novocastra (Leica Biosystems Newcastle Ltd., UK) and diluted 1:100. A polyclonal site-directed antibody against δ-SG was prepared using a synthetic peptide from the sequence of the cloned δ-SG cDNA and diluted 1:1000 [23 (link)]. A monoclonal mouse antibody against the α-spectrin chain (clone AA6) was purchased from Merck Millipore (Germany) and used at a 1:200 dilution. A monoclonal anti-α-tubulin antibody (clone DM 1A) was purchased from Sigma-Aldrich (USA) and used at a 1:10,000 dilution. For immunofluorescence microscopy, Alexa Fluor® 488- or 594-labeled secondary antibodies were obtained from Molecular Probes® (Life Technologies) and diluted to 1:500 before use.

Free-floating sections were incubated in 80% methanol for 20 min at RT. Sections were blocked for 1 h at RT and then incubated overnight at 4 °C with rabbit anti-Iba1 (1:1000) and goat anti-CX3CR1 (1:50) or mouse anti-NLRP3 (1:300) primary antibody in the blocking buffer. Sections were then incubated for 1 h at RT with appropriate Alexa Fluor 488- or 594-labeled secondary antibodies (1:1000, Molecular Probes, Eugene, OR, USA). After processing for 30 s with TrueBlack solution (Biotium, Hayward, CA, USA) to quench lipofuscin autofluorescence, sections were coverslipped with VECTASHIELD (Vector Laboratories). Images of double-stained cells in the dentate gyrus were taken at ×400 magnification with a fluorescence microscope (Olympus BX-41) using cellSens imaging software (Olympus Corporation).