Ab11959

Protein was extracted from the platelets or brain homogenates using RIPA buffer supplemented with a protease inhibitor and quantified using a bicinchoninic acid (BCA) (23,227, Thermo, USA) kit. Equal amounts of protein from each sample were separated using 8% SDS-PAGE, transferred onto a nitrocellulose membrane, and incubated overnight with antibodies targeting p-RIP1 (Ser166, 1:1000, 53,286, CST, USA), RIP1 (1:1000, ab202985, Abcam, USA), p-RIP3 (Ser232, 1:1000, ab195117, Abcam, USA), RIP3 (1:1000, ab62344, Abcam, USA), p-AKT (Ser 473, 1:1000, 4060 T, CST, USA), AKT (1:1000, 4691S, CST, USA), Fosb ( 1:1000, ab11959, Abcam, USA), Jun (1:1000, ab40766, Abcam, USA), Jund (1:1000, ab181615, Abcam, USA), Fos (1:1000, ab222699, Abcam, USA), Junb (1:1000, 128,878, Abcam, USA), and β-actin (1:5000, A5441, Sigma) in Tris buffered saline containing 0.2% Tween-20 (TBST) and 5% nonfat dry milk at 4 °C. After washing with TBST, the membranes were incubated with 1 μg/ml goat anti-rabbit IRDye 800CW or goat anti-mouse IRDye 800CW (Licor Odyssey, USA). The positive bands were detected using the Odyssey infrared imaging system (LI-COR Biosciences, USA), and signal intensity was quantified using ImageJ software and normalized to that of β-actin.

Take out the dehydrated brain, cut it perpendicularly to the missing seam, place it on the stage, and embed it in OCT. Using a cryostat (Leica CM1950), put it in a microtome and quickly freeze it at −20°C for 20 min. Then transfer it to the slicing table, perform coronal sectioning with a thickness of 30 um, gently pick it out with a brush, and place it in a six-well plate of 0.1 M PBS. Three sets of brain slices were selected for each group, and rinsed for three times at room temperature with 0.1MPBS (pH 7.4) for 10 min each time. Then place the brain slices in the antibody diluent (3% BSA, 0.3% Triton X-100 PBS) that has been added to the primary antibody, and incubate at room temperature for 16 h on a shaker. Use the following primary antibodies: mouse anti c-fos (1:500; abcam, ab11959), rabbit anti CaMKII (1:200; abcam, ab5683). The brain slices were rinsed three times with 0.1MPBS at room temperature for 10 min each time, and then the brain slices were placed in the antibody diluent with the added secondary antibody, incubated for 4 h, and DAPI was added at 3.5 h. Use the following secondary antibodies: Donkey Anti-Rabbit IgG H&L Alexa Fluor® 488 (1:500; invitrogen, A-21206), Goat Anti-Mouse IgG H&L Alexa Fluor® 594 (1:500; invitrogen, A-21202), DAPI (1:1000; Sigma, D9542). Then the brain slices were rinsed for three times with 0.1MPBS for 10 min each time.

Immunofluorescent histochemical procedure was applied to evaluate the double-labeling of FOS/CaMKII and FOS/GAD67 in the IC of sham and SNI mice as described in our previous study (Zhang et al., 2022 (link); Zhu et al., 2022 (link)). Briefly, mice were perfused with 0.1 mol/L PBS and 4% paraformaldehyde for fixation, and then serially cut into transverse slices with 30 μm thickness. All serial sections were then incubated with primary antisera (1:400, ab11959, Abcam, MA, United Kingdom) for 18–24 h at 4°C in 0.01 M PBS containing 1% (v/v) normal donkey serum, 0.3% (v/v) Triton X-100, 0.02% (w/v) sodium azide, and 0.12% (w/v) carrageenan (pH 7.4). Then, the sections were incubated with Alexa 488 donkey anti-rabbit (1:500, A21206, Invitrogen)/Alexa 594 donkey anti-mouse (1:500, A21203, Invitrogen, CA), and Alexa 488 donkey anti-mouse (1:500, A21202, Invitrogen)/Alexa 594 donkey anti-rabbit (1:500, A21207, Invitrogen) for 6–8 h at 4°C. If necessary, the sections were incubated with tertiary antisera for 2–4 h in 0.01 m PBS with 0.3% (v/v) Triton X-100 at 4°C. After the immunofluorescence histochemical staining, the sections were observed and images were captured using VS200 microscope (VS200, Olympus, Japan). Digital images were captured using VS200 software (Olympus).