K3464

Placental samples were taken after delivery. Several placentomes and the inter-cotyledonary chorion were fixed in 10% buffered formalin, processed by standard methods, and embedded in paraffin-wax blocks (29 (link)). About 4 μm-thick sections of tissue were cut, mounted on glass microscope slides, and stained with hematoxylin and eosin (H&E) and examined by microscopy (Olympus, Japan).
Tissues with N. caninum-compatible lesions were selected and analyzed by immunohistochemistry (IHC) as previously described by Campero et al. (29 (link)) with minor modifications. An Avidin-Biotin commercial kit (ABC Elite ABC Peroxidase Complex Vector PK6101, Vector, Burlingame, United States) was employed according to the instructions of the manufacturer. A rabbit polyclonal antibody against NC-1 tachyzoites was used as the primary antibody at 1: 300 dilution for an incubation period of 45 min at 37°C (kindly provided by Dr. Mark Anderson, UCDavis, Davis, United States). Secondary antibody (provided by the kit) was incubated for 30 min at 37°C and AEC substrate chromogen K3464 (Dako, Carpinteria, CA, United States) was incubated for 5 min at room temperature. Finally, sections were counterstained for 5 min with Mayer hematoxylin and rinsed with distilled water.

Immunohistochemical staining was performed as described previously (21). Briefly, the tissue sections were boiled in citrate buffer (Dako target retrieval solution, S1699, DAKO, Carpenteria, CA) to achieve antigen retrieval for 20 min. The slides were then incubated with peroxidase blocking solution (S2023, DAKO) for 15 min and protein block serum-free (X0909, DAKO) for 30 min. Tissue sections were incubated overnight at 4 C with primary antibodies: type I collagen (1:200; 1310-01; Southern Biotech, Birmingham, AL, USA), intercellular adhesion molecule-1 (ICAM-1) (1:200; sc1511; Santa Cruz Biotechnology, Santa Cruz, CA, USA), F4/80 (1:200; 14-4801-81; eBioscience, San Diego, CA, USA) and fibroblast-specific protein 1 (FSP-1)/S100A4 (1:200; ab41532; Abcam, Cambridge, UK). Second antibodies were incubated for 30 min at room temperature and then treated with horseradish peroxidase-conjugated streptavidin (P0397, DAKO) for 30 min. To visualize the immunocomplexes, the testicular sections were treated with 3-amino-9-ethyl carbazole substrate solution (K3464, DAKO). The F4/80-positive macrophages and FSP1-positive fibroblasts per high-power field were counted at a magnification of $\times$ 200. The ImageJ software was used to measure the fibrotic areas of type I collagen and ICAM-1 positive area in 10 randomly chosen non-overlapping fields at a magnification of $\times$ 200.