Double-labeling Immunofluorescence Analysis

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).