Histopathological Analysis of Liver and Spleen in Parasite Infection

Livers and spleens were fixed in 10% neutral phosphate buffered formalin. The paraffin-embedded organs were cut into 4 μm-thick sections, followed by hematoxylin and eosin staining for light microscopy. For the detection of parasites, liver sections were performed by indirect immunostaining using human serum infected with L. martiniquensis (1 (link)) (1:500 dilution) and peroxidase-conjugated AffiniPure goat anti-human IgG heavy and light chain antibody (1:500 dilution; Jackson ImmunoResearch, PA, USA). The peroxidase activity was visualized using a solution of 3,3'-diaminobenzidine (DAB) (Wako, Tokyo, Japan) and H₂O₂ (pH 7.0) for 4 min. The sections were washed in distilled water and counterstained with Mayer's hematoxylin before dehydration and mounting.
The cell-mediated immune response (CMIR) of the liver against parasitized Kupffer cells was classified into no granuloma, immature granuloma, mature granuloma, and involuting granuloma (22 (link), 23 (link)). The number of each response was counted in 25 consecutive microscopic fields per mouse at ×400 magnification. The histopathological reaction and CMIR of the spleen were determined, according to previously published protocols (24 (link)–27 (link)).