Goat anti rat igg rhodamine

Animals were anaesthetized and perfused intracardially with 30 ml ice-cold PBS and 30 ml of 4% paraformaldehyde. Spinal cords were removed and soaked in 30% sucrose/PBS for 3 days. Frozen spinal cord sections (cervical, thoracic, lumber, and sacral regions) were cut at a thickness of 20 µm by cryostat and stained with myelin basic protein (MBP) and neurofilament primary monoclonal antibodies (Abcam) and visualized with fluorescent secondary antibodies for MBP (Goat anti-Rat IgG FITC) and neurofilament (Goat anti-Rat IgG Rhodamine) (Jackson Immunoresearch Laboratories, Inc.). Cell nuclei were counterstained with DAPI for the analysis of total cellular infiltration. Digital images of spinal cord sections were taken with an Axiovert 200 (Zeiss) microscope. Areas of extensive cellular infiltration, demyelination, and axonal loss were manually traced with the AxioVision imaging system onto spinal cord images. Then, the total areas of cellular infiltration, demyelination, and axonal loss in spinal cord sections were calculated by the AxioVision imaging system. The percentage of lesions was calculated as (lesion area/total spinal cord area) ×100.

MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK) was purchased from Protein and Nucleic Acid Core Facility Stanford University, Stanford, CA, USA. Pertussis toxin was purchased from List Biologicals, St. Louis, MO, USA. Methocel was purchased from Sigma Aldrich, St. Louis, MO, USA. Rabbit anti-MBP Ab, rat anti-neurofilament Ab, rabbit Anti-Ym-1 Ab, rabbit anti-iNOS Ab for immunohistology were purchased from Abcam, Cambridge, MA, USA. Rat anti-CD68 Ab was purchased from Biorad, Hercules, CA, USA. Mouse anti-GFAP-Alexa fluor 488 Ab was purchased from Biolegend, San Diego, CA, USA. Rat anti-C3 Ab was purchased from Novus Biologicals, Littleton, CO, USA.
Donkey anti-rabbit IgG Alexa 488, goat anti-rat IgG Rhodamine, goat anti-rabbit IgG Rhodamine, and chicken anti-rat IgG Alexa488 were purchased from Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA. Antibodies against F4/80, iNOS, and CD206 for flow cytometry and ELISA kits for IFN-γ, IL-17A, GM-CSF and IL-10 were purchased from eBioscience, San Diego, CA, USA.