gondii in sheep ( Pereira-Bueno et al., 2004 and Motta et al., 2008). Considering that the consumption of ovine meat occurs in different countries around the world, the aim of this study was to identify T. gondii by IHC in different sheep tissues and to determine if an association exists between the results obtained by this method and those obtained by the MAT. This study was approved by

the Ethics Committee of Animal Use (CEUA) from the Universidade Federal Fluminense (UFF) under protocol number 00111/09. Tissue samples were collected from 26 seropositive sheep with different titres for T. gondii by MAT, after the slaughter of the animals. These sheep belonged to a larger group of 287 animals that had been previously tested for the parasite by MAT in despite of the titres that they

presented. At the time of the study, only OSI-744 in vitro these 26 sheep were allowed by the owners to be slaughtered. The samples were submitted to histopathological evaluation and identification of the parasite by IHC. The serological analysis was performed with the MAT according to Dubey and Desmonts (1987). All samples with agglutinating activity at a dilution of selleck chemicals 1:25 were considered positive (Sousa et al., 2009). These serum samples were subsequently titrated against reacting antigens using serial two-fold dilutions up to 1:3200. Tissue specimens from liver, heart, brain, diaphragm, kidney and lung were collected from 26 T. gondii-seropositive sheep and fixed in neutral-buffered, 10% formalin. These specimens were routinely processed in paraffin for light microscopy and histological sections were produced for both haematoxylin–eosin (H&E) and IHC staining. The presence of T. gondii tissue cysts was investigated in IHC-stained sections of the brain, heart and liver of 26 seropositive sheep. The histological sections were deparaffinised

and hydrated, and the endogenous peroxidase was blocked with a 3% hydrogen peroxide solution. The sections were incubated in a 96 °C water bath for 30 min for antigen recovery. The nonspecific binding was blocked by incubating the sections in a solution of milk and 10% bovine serum albumin for 30 min. Subsequently, the sections were incubated for 30 min with primary rabbit anti-T. gondii antibody (Neomarkers, Fremont, CA, USA) diluted 1:200. The sections were Cell press treated with DAKO LSAB DAKO Corp. Carpinteria, CA, USA) as recommended by the manufacturer. Diaminobenzidine (DAB; DAKO Corporation, Carpinteria, CA, USA) was used as the chromogen to reveal the life cycle stages of the parasite, and all samples were counterstained with Harris haematoxylin. Histological sections of human brain positive for T. gondii were used as positive controls for the IHC technique as recommended by the manufacturer, and the primary antibody was omitted for negative controls. The samples were considered positive when bradyzoite pseudocysts were stained in brown by DAB.