EAE induction and pathological analyses
To identify associations between PRXs and CNS inflammation, we isolated the spinal cords from EAE mice for pathological analysis. EAE was induced in mice using the same method described in our previous study [16]. Simply, a total of 200 μg myelin oligodendrocyte glycoprotein peptide 35–55 in complete Freund’s adjuvant containing killed Mycobacterium tuberculosis H37Ra was subcutaneously administered to wild-type C57BL/6 mice (10 weeks old, female) (day 1). Then, the mice received intraperitoneal injections of 400 ng pertussis toxin (days 1 and 2). EAE mice were scored using the following scale: 0.0 = no clinical signs, 1.0 = partial paralysis of the tail, 2.0 = limp tail and mild bilateral hind leg paralysis, 3.0 = limp tail and complete paralysis of the hind legs, 4.0 = limp tail and complete hind leg and partial front leg paralysis, and 5.0 = complete hind and front leg paralysis. In this study, we used spinal cords from EAE mice (n = 2) whose score was 4.0 on day 18 to investigate the role of PRXs in the established inflammatory CNS lesions. Untreated normal (naive) mice (n = 2) were used as controls. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Chiba University (Approved No. 1-9).
Histopathological examinations were performed using paraffin-embedded sections of spinal cords, and the sections were stained with hematoxylin and eosin (HE), mouse anti-glial fibrillary acidic protein (GFAP) (Novocastra, NCL-GFAP-GA5), rabbit anti-PRX1 (ProteinTech Group, 15816-1-AP), rabbit anti-PRX2 (ProteinTech Group, 10545-2-AP), rabbit anti-PRX3 (ProteinTech Group, 10664-1-AP), rabbit anti-PRX4 (ProteinTech Group, 10703-1-AP), rabbit anti-PRX5 (ProteinTech Group, 17724-1-AP), rabbit anti-PRX6 (ProteinTech Group, 13585-1-AP), rat anti-CD3 (Abcam, ab56313), and rat anti-CD45 (Santa Cruz Biotechnology, SC-53665). Alexa Fluor594 chicken anti-rabbit IgG (Invitrogen, A21442) and goat anti-rat secondary antibody Alexa Fluor488 (ThermoFisher, A-11006) were used as secondary antibodies.