Cultures were fixed in freshly prepared, buffered 4% paraformaldehyde. After blocking with 20% normal goat serum and permeabilization for 10min with 0.2% Triton X-100 in PBS, cultures were incubated overnight at 4°C with the following antibodies (mAb, monoclonal; pAb, polyclonal): Nestin ( MAB5326, Millipore, 1:200), Ki67 (ab16667, Abcam, 1:1000), Oct4 (MAB4401, Millipore, 1:2000), Pax6 (Developmental Studies Hybridoma Bank; 1:30), β-tubulin isotype III (β-tubIII, mAb, MMS-435P Covance, 1:400), Glial Fibrillary Acidic Protein (GFAP, z033429-2, Dako, 1:400), Lamp1 (pAb, ab24170, Abcam, 1:750), Cleaved Caspase-3 (pAb, #9661, Cell Signaling, 1:500), NLRP3 (pAb, AG-20B-0014-C100, AdipoGen, 1:200), Microtubule-Associated Protein 2 (MAP2, AB5622, Millipore, 1:400), Caspase 1 (Casp1, SC-515, Santa Cruz Biotechnology, 1:100), LC3B (pAb#2775, Cell Signaling, 1:400), GAPDH (ab9485, Abcam, 1:3000). After removal of the primary antibodies and repeated washes with PBS, cultures were incubated at room temperature for 45 min with secondary antibodies labeled with Alexa Fluor 633 or 488 (anti-mouse and/or anti-rabbit, Molecular Probes). Samples were then labeled with DAPI (0.3 μg/ml, Roche) for nuclear staining and rinsed with PBS for mounting and analysis. Microphotographs were taken using a confocal microscope.
