Rabbit anti cleaved caspase 1

For immunofluorescence staining, the cultured neurons (on silicone membrane) and brain tissue sections (10-μm thickness) were washed with 1× PBS and fixed in 4 % paraformaldehyde for 20 min at 25 °C. After fixation, the washed membrane or tissue sections was then blocked with 3 % normal goat serum at 25 °C for 1 h in the presence of 0.1 % Triton X-100. The cell culture membranes or tissue sections were incubated overnight at 4 °C with primary antibodies to rabbit anti-cleaved caspase-1 (Thermo scientific) and mouse anti-NeuN (Abcam). All antibodies were used at the concentration of 3 μg/mL. After washing with PBS, cells or tissue sections were incubated with Alexa Fluor 488 or 594 conjugated with anti-mouse or anti-rabbit immunoglobulin G (IgG) for 1 h and mounted with immunomount containing DAPI (Invitrogen) on a slide. The silicone membrane was mounted on a cover slip and carefully cut at the edges using a scalpel blade and then again mounted on a slide using DAPI. The photographs were captured using fluorescent microscope Eclipse TE2000-U (Nikon, Melville, NY) using the NIS Elements software (Nikon, Melville, NY). Controls for the experiments consisted of the omission of primary antibodies. No staining was observed in these cases. The intensity of immunostaining was analyzed by ImageJ (NIH) software.

Primary antibodies mouse anti-NeuN and rabbit anti-IL-1β were purchased from Abcam (Cambridge, MA), mouse anti-β-actin and rabbit anti-cleaved caspase-1 from Thermo scientific (Rockford, IL), and rabbit anti-PARP from BD Biosciences (San Jose, CA). All secondary Alexa Fluor-conjugated antibodies, Fluo 4AM, TUNEL kit, and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). ELISA kit for IL-1β was purchased from Thermo Scientific. 3,3′-diaminobenzidine (DAB) was purchased from Sigma-Aldrich (St. Louis, MO); z-YVAD-fmk (caspase-1 inhibitor) was from Santa Cruz Biotechnology (Dallas, TX); and tetrodotoxin (TTX) was from Cayman chemicals (Ann Arbor, MI).