Rabbit anti ptaok2

According to our previous report,⁶³ (link) after being deeply anesthetized with pentobarbital sodium, rats were transcardially perfused with 150 mL of 1× PBS (4°C) followed by 150 mL of 4% paraformaldehyde (4°C). Lumbar enlargement of the spinal cord was collected, postfixed in 4% paraformaldehyde for 48 h at 4°C, and subsequently dehydrated with 30% sucrose for 3 days at 4°C. Subsequently, the spinal cord was cut transversely into slices with a thickness of 30 μm. The sections were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 10% sheep or donkey serum for 2 h at 24°C. Sections were incubated with the following primary antibodies for 48 h at 4°C: goat-anti-Iba1 (1:200, Abcam, Cambridge, UK), mouse anti-GFAP (1:500, Cell Signaling Technology), mouse-anti-NeuN (1:500, Abcam), rabbit-anti-pTAOK2 (1:100, GeneTex), rabbit-anti-cGAS (1:100, Cell Signaling Technology), and rabbit-anti-STING (1:100, Cell Signaling Technology). After washing with 1× PBS, the sections were incubated with fluorescent secondary antibodies in the dark for 2 h at 24°C. Finally, the sections were examined under a fluorescence microscope (FV3000; Olympus, Japan).

The Western blot protocols were performed according to our previous studies.⁶² (link) After deep anesthesia with pentobarbital sodium, rats were decapitated, and lumbar enlargement of the spinal cord was quickly harvested and divided into four parts. The dorsal horns were selected and homogenized on ice using a lysis buffer containing protease and phosphatase inhibitors. The tissue homogenates were separated by 10% SDS-PAGE and subsequently transferred to 0.45-μm PVDF membranes. The membranes were blocked with 5% skimmed milk at 24°C for 1 h. Thereafter, membranes were incubated with the following primary antibodies for 24 h at 4°C: rabbit-anti-TAOK2 (1:500; GeneTex, TX, USA), rabbit-anti-pTAOK2 (1:1000, GeneTex), rabbit-anti-cGAS (1:1000, Cell Signaling Technology, Mass, USA), rabbit-anti-STING (1:500, Cell Signaling Technology), mouse-anti-tubulin (1:2000, Beyotime Biotechnology, Shanghai, China), and mouse-anti-GAPDH (1:10,000, Proteintech, IL, USA). After washing the primary antibody in TBST, the membrane was incubated with HRP-conjugated secondary antibody for 2 h at 24°C. The ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) was used to detect the immune complex bands.