Rat anti cd4

After beam walking test, mice were sacrificed by pentobarbital overdose and received intracardiac perfusion with ice-cold PBS and paraformaldehyde. Cervical spinal cords were harvested, and sectioned at 10-μm thickness with a cryostat (Thermo Fisher Scientific, USA). Immunofluorescence was performed with standard procedure. The following primary antibodies were used: (1) rabbit anti-human IgG (1:600, Dako, USA), (2) rabbit anti-AQP4 (1:200, Sigma-Aldrich, USA), (3) mouse anti-glial fibrillary acidic protein (GFAP, 1:200, Santa Cruz Biotechnology, USA), (4) goat anti-myelin basic protein (MBP, 1:200, Dako, USA), (5) rabbit anti-neurofilament heavy polypeptide (NF-H, 1:400, Sigma-Aldrich, USA), (6) rabbit anti-NR2B (1:200, Abcam, UK), (7) rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1, 1:200, Wako, Japan), (8) rat anti-lymphocyte antigen 6 complex locus G6D (Ly6G, 1:400, Abcam, UK), (9) rat anti-CD4 (1:100, Santa Cruz Biotechnology, USA), and (10) rat anti-CD8 (1:100, Santa Cruz Biotechnology, USA). Sections were then incubated with the appropriate Alexa-Fluor-conjugated secondary antibodies (Thermo Fisher Scientific, USA) at room temperature for 1 h. They were counterstained with DAPI and mounted with anti-fade reagent (Thermo Fisher Scientific, USA).

Paraffin-embedded intestinal tissue sections were used for immunohistochemistry. Tight junction proteins ZO-1 and Occludin expression was detected using rabbit anti-ZO-1 (1:1000, Abcam, Cambridge, MA, USA) and rabbit anti-Occludin antibodies (1:50, Abcam, Cambridge, MA, USA), respectively. Thereafter, the sections were immunostained with goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) using a DAB kit (Zhongshan Goldenbridge, Beijing, China).
T-cell markers CD3, CD4, CD8a, and CD25 were detected using mouse anti-CD3, rat anti-CD4, mouse anti-CD8a and mouse anti-CD25 (1:250, Santa Cruz Biotechnologies, Dallas, TX, USA), respectively. For the secondary antibody, we utilized goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) using a DAB kit (Vector Laboratories, Inc., Burlingame, CA, USA).