Ab3683

The expression of MPO was detected by IF staining. Paraffin‐embedded tissue sections (5 mm thickness) were dewaxed in xylene, then dehydrated using graded concentrations of alcohol, and incubated with 3% H₂O₂ to inhibit endogenous peroxidase. After being blocked in 10% goat serum for 10 minutes at room temperature, they were incubated with the primary antibody (anti‐MPO 1:200, ab3683, Abcam) in a blocking solution overnight at 4°C. After slides were washed in PBS, HRP‐labeled anti‐rabbit IgG (1:100; AS014, ABclonal) was applied for 30 minutes at 37°C, and then washed with PBS again. Then, images were obtained with a Nikon EclipseNi inverted microscope (TE2000, Nikon).

Western blotting was in accordance with the description in our previous study [22 (link)]. Briefly, L4–L6 spinal dorsal horn tissues of animals were removed and homogenized in 15 mmol/l Tris containing a cocktail of proteinase inhibitors and phosphatase inhibitors following animals were anesthetized with 50 mg/kg sodium pentobarbital (i.p.). Next, the lysates of L4–L6 dorsal horn tissues were prepared and separated by gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane. They were then pre-incubated for 1 h at room temperature in the block buffer. After incubating with diluted primary antibodies against SIRT1 (1:2000, #ab110304, Abcam, UK), NALP1 (1:1000, #ab3683, Abcam, UK), phosphorylated STAT3 (1:1500, #ab76315, Abcam, UK), acetylated histone H3 (1:1000, #ab8898, Abcam, UK), acetylated histone H4 (1:1000, #07–329, Millipore, USA), or β-actin (1:2000, #ab8226, Abcam, UK) overnight at 4 °C, the membranes were incubated in horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the bands in the membranes were visualized by enhanced chemiluminescence (ECL, Pierce, USA) as directed by the manufacturer. The bands then were quantified with computer-assisted imaging analysis system (NIH ImageJ).