Asr1651

After fixation with 4% paraformaldehyde, tissue paraffin 4 μm slices were conventional dyed with hematoxylin and eosin (H&E) and Nissl staining and immunohistochemistry were conducted to analysis the histopathological condition around the injury site. Nissl staining was conducted by Nissl Staining Solution (Cresyl Violet) (Solarbio Science & Technology Co., Ltd., Beijing, China). The brain sections were incubated with Nlrp3 antibody (ab270449, Abcam, 1:100) overnight at 4°C, incubated with HRP-linked secondary antibody (ASR1651, Abcepta), and stained by DAB solution. Microscopic observation of the histological slides was performed using a light microscope (10× objective). Both Nissl-positive cells in randomly three fields of primary somatosensory cortex around the injury site (parietal cortex) per brain were counted by the ImageJ software (1.4, NIH).

After lysed, the protein concentration was quantified using a bicinchoninic acid protein assay kit (Thermo Scientific). 30 μg protein per lane were loaded on SDS-PAGE gels. After electrophoresis, the proteins were transferred to PVDF membranes. The membranes were blocked with 5% BSA and incubated at 4°C overnight with the appropriate primary antibodies: IbA1(17198S, CST, 1:1,000), Nlrp3 (ab270449, Abcam, 1:1,000), caspase1 (ab179515, Abcam, 1:1,000), caspase1 (p20) (AG-20B-0042-C100, AdipoGen, 1:1,000), Il-1β (31202S, CST, 1:1,000), Gapdh (YM3029, Immunoway,1:5,000), and incubated with horseradish peroxidase-conjugated secondary antibodies (ASR1937 and ASR1651, Abcepta, 1:20,000). Protein bands were visualized by enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA) and images were captured using a ChemiDoc™ MP imaging system (Bio-Rad). All samples were run in parallel with four replicates.