Nlrp3 nbp2 12446

Under pentobarbital sodium (50 mg/kg) anesthesia, the rats were perfused with normal saline and then L6–S1 spinal cord was harvested. Tissue specimens were fixed with 4% paraformaldehyde and then dehydrated with 30% sucrose. After embedding with optimal cutting temperature compound, tissue specimens were sectioned at 20 μm thickness. Sections were blocked with 5% bovine serum albumin and incubated with primary antibodies including NLRP3 (NBP2-12446, 1:100; Novus Biologicals, USA), NeuN (MAB377, 1:200; Millipore, USA), GFAP (3670, 1:300; Cell Signaling Technology, USA) and OX-42 (MCA275G, 1:100; Bio-Rad, USA). Then sections were incubated with secondary antibodies conjugated with Cy3 or FITC, and mounted with antifade mountants (containing DAPI). Images were captured using an inverted fluorescence microscope (EVOS FL, Thermo Fisher Scientific, USA) and fluorescent colocation was quantified by Image J software (National Institutes of Health, Bethesda, USA).

Regenerative and inflammatory markers were analyzed using optic nerve tissue. Lysates were produced from optic nerve tissue using a PRO-PREP solution (iNtRON Biotechnology, Gyeonggido, Korea). The same amounts of total proteins were separated by SDS-electrophoresis and transferred to the membrane. The membranes were incubated with anti-Thy-1 (SC-53116), β-actin (SC-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Vegf (GTX102643), Tnf-α (GTX10520), β-catenin (GTX101435), Wnt3a (GTX128101), (GeneTex, Irvine, CA, USA), or GFAP (#3670), Neurofilaments (#2837), tCaspase3 (#9662), Bcl2 (#2764), Nf-κb (#8242) (Cell Signaling Technology, Danvers, MA, USA) or Hif-1α (PA1-16601), Bdnf (PA5-85730), Iba1 (PA5-27436) (Thermo Fisher Scientific) or Nlrp3 (NBP2-12446) (Novus Biologicals, Centennial, CO, USA) or NeuN (MABB377) (Millipore) antibodies. All antibodies except Thy-1 (1:200 dilution) were used in a 1:1000 dilution ratio. After washing steps, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or mouse secondary antibodies at a 1:10,000 dilution (GeneTex) for o/n at 4 °C. Immuno-active bands were visualized as enhanced chemiluminescence solutions (Bio-Rad Laboratories, Hercules, CA, USA) and detected using ImageQuant™ LAS 4000 (GE Healthcare, Chicago, IL, USA).