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.