An immunohistochemical examination using anti-T. equiperdum rabbit
antisera (1:400, K. Suganuma) as the primary antibody was performed to evaluate the
distribution of parasites in each organ and tissue. In addition, to identify the phenotype
of inflammatory cells, selected sections were immunostained with anti-CD3 rabbit
monoclonal antibody (clone SP7, 1:400, Abcam, Cambridge, UK) and anti-Iba1 rabbit
polyclonal antibody (1:500, Wako, Osaka, Japan). Sections were deparaffinized by xylene
and hydrated in a series of graded ethanol. Non-specific endogenous peroxidase was blocked
with 0.3% H2O2 at room temperature for 10 min. Sections were
incubated with-each primary antibody at 4°C overnight. MAX-PO polymer reagent (Nichirei,
Bioscience, Tokyo, Japan) was used as the secondary antibody at room temperature for 30
min. Labeling was visualized by 3,3′-diaminobenzidine, and sections were counterstained by
Mayer’s hematoxylin. Additionally, selected sections were applied for immunofluorescence
for T. equiperdum. In immunofluorescence, Alexa Fluor 488-conjugated goat
anti-rabbit IgG (1:400, Thermo Fisher Scientific, Hanover Park, IL, USA) was used for
secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Vector
Laboratories, Burlingame, CA, USA).
antisera (1:400, K. Suganuma) as the primary antibody was performed to evaluate the
distribution of parasites in each organ and tissue. In addition, to identify the phenotype
of inflammatory cells, selected sections were immunostained with anti-CD3 rabbit
monoclonal antibody (clone SP7, 1:400, Abcam, Cambridge, UK) and anti-Iba1 rabbit
polyclonal antibody (1:500, Wako, Osaka, Japan). Sections were deparaffinized by xylene
and hydrated in a series of graded ethanol. Non-specific endogenous peroxidase was blocked
with 0.3% H2O2 at room temperature for 10 min. Sections were
incubated with-each primary antibody at 4°C overnight. MAX-PO polymer reagent (Nichirei,
Bioscience, Tokyo, Japan) was used as the secondary antibody at room temperature for 30
min. Labeling was visualized by 3,3′-diaminobenzidine, and sections were counterstained by
Mayer’s hematoxylin. Additionally, selected sections were applied for immunofluorescence
for T. equiperdum. In immunofluorescence, Alexa Fluor 488-conjugated goat
anti-rabbit IgG (1:400, Thermo Fisher Scientific, Hanover Park, IL, USA) was used for
secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Vector
Laboratories, Burlingame, CA, USA).