Anti neun

Mice were sacrificed at the third day after zinc administration. The tissue was prepared as described above. The frozen sections were dried at room temperature for 2 hours. The sections were then incubated at room temperature with 5% normal goat serum containing 0.3% Triton X‐100 for 2 hours. Then, 1x PBS was used to wash the section three times for 5 minutes each. Primary antibody was added, and the sections were incubated with gentle shaking for 12 hours at 4°C. On the following day, 1x PBS was used to wash the section three times for 5 minutes each. Next, the sections were incubated with secondary antibody for 8 hours on a shaker at 4°C. 4'6‐Diamidino‐2‐phenylindole (DAPI) solution was used to stain the nuclei. The slices were selected from 0.5 cm‐long pieces of tissue from above and below the epicenter of the damaged spinal cord. For each mouse, select every other continuous section and count three sections in total. In the ventral horn area on both sides of the spinal cord, a 200 × 200 µm area was selected to count the number of neuron‐positive cell, IBA‐1‐positive cell, and NLRP3‐positive cell. The antibodies used are listed as followed: anti‐NeuN (1:1000; abcam, UK), anti‐Iba‐1 (1:500; Wako, JP), and anti‐NLRP3 (1:250, Affinity, USA).

Immunofluorescent staining was performed according to the procedure published previously^{31 (link)}. For mouse brain tissue staining, 25 μm-thickness sections were cut by using Cryostat (Leica). The following antibodies were used: anti-GFAP (1:300, Agilent Technologies, Z0334), anti-NeuN (1:500, Abcam, ab177487) and anti-CD68 (1:200, BioRad, MCA1957). Brain sections were incubated in the primary antibody overnight at 4 °C followed by incubation in the secondary antibody for 2 hours at room temperature. The images were captured by using the FLUOVIEW FV10i confocal microscope (Olympus Life Science) and quantified with ImageJ.

The following primary antibodies and dilutions were used for Immunostaining and Western blotting anti‐Arid1a (Sigma‐Aldrich, HPA005456); anti‐biotinylated IsolectinB4 (Vector Laboratories, B‐1205); anti‐GFAP (Dako, Z0334); anti‐GFAP (Sigma, G6171); anti‐SOX2 (Cell Signalling Technology, 3728s); anti‐Tbr1 (Abcam; ab31940); anti‐GLAST (Proteintech, 20785‐1‐AP); anti‐S100𝛽 (Abcam, ab52642); anti‐MAP2 (Millipore, MAB3418); anti‐BLBP (Abcam, ab32423); anti‐PDGFR𝛽 (Abcam, ab32570); anti‐CTIP2 (Abcam, ab18465); anti‐SATB2 (Abcam, ab51502); anti‐TUJ1 (Sigma, T2200); anti‐NeuN (Abcam; ab177487); anti‐𝛽‐Actin (Proteintech, 20536‐1‐AP); anti‐𝛽‐Actin (Proteintech; 60008‐1‐Ig); anti‐CD31(BD Biosciences, 553370); anti‐IgG (Bioss; bs‐0295p); anti‐Flag (Sigma, F1804), anti‐HA (Cell Signaling Technology); anti‐Claudin 5 (Invitrogen, 35‐2500). The following florescence secondary antibodies were used: anti‐rabbit Cy3 (Jackson ImmunoResearch), anti‐rat Cy3 (Jackson ImmunoResearch), anti‐mouse Cy3 (Jackson ImmunoResearch), anti‐rat Alexa Fluor 488 (Jackson ImmunoResearch), anti‐rabbit Alexa Fluor 488 (Jackson ImmunoResearch), anti‐goat Alexa Fluor 488 (Jackson ImmunoResearch).