Vs200

Fresh-frozen human brain tissues were acclimated to room temperature and then exposed to post-fixation by 4% paraformaldehyde. Following a 10 min wash in buffer (TBS containing 0.1% Triton-X), tissues were exposed to protein block (5% goat serum, 0.1% Triton-X 100, 1% BSA; in TBS). Tissues were incubated with rabbit anti-PBR (ab109497; Abcam, Cambridge, MA). After washing, sections were incubated with a rabbit-on-rodent polymer (Vector Laboratories, 30 min) secondary antibody and detected with 3,3′-diaminobenzidine development. Sections were counter-stained with haemotoxylin, dehydrated and coverslipped. Slides were imaged using an Olympus VS200 (Olympus Corporation).

Colons from mice were harvested and fixed with a 4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako). Sections obtained from fixed specimens were permeabilized and incubated with an anti-H. cinaedi antibody (1:1,000) as the first antibody at room temperature for 1 h. The anti-H. cinaedi antibody was obtained by immunizing rabbits with heat-inactivated H. cinaedi MRY08-1234 as an antigen, followed by protein A-purification. The sections were then rinsed and stained with Alexa Fluor 488 (H. cinaedi) and 4′,6-diamidino-2-phenylindole (nuclei). Fluorescence signals were visualized using an automated slide scanning system (Olympus VS200, Olympus, Tokyo, Japan), and images were analyzed using ImageJ version 1.53t.

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).

Immunofluorescent histochemical procedure was applied to evaluate the double-labeling of CaMKII/FOS and GAD67/FOS in the ACC of sham and SNI mice. 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). Image J (NIH Image) software was utilized to count the number of double labeled neurons using.

We performed Sholl analysis for glial cell morphology as described previously [17 (link)]. In brief, images at high resolutions were obtained with OLYMPUS VS200 after immunofluorescent staining. Sholl analysis was performed with the plugin of Sholl analysis in ImageJ that automatically draws a series of concentric circles at 10-μm intervals from the center of DAPI signal to the end of the most distant process in every single cell. The number of intercepts of GFAP or ionized calcium-binding adapter molecule (Iba1) positive processes in each circle, ending radius, and ramification index were analyzed. A total of 20 cells were analyzed in each group.