**2.4 Characterization of** *T. gondii* **stages by immunolabeling**

The differentiation of parasites in culture cells infected with bradyzoite forms of *T. gondii* was monitored using tachyzoite stage-specific anti-SAG-1 antibodies (kindly provided by Dr. José Roberto Mineo - Immunoparasitology Laboratory, Federal University of Uberlândia, Minas Gerais, Brazil). Bradyzoites of *T. gondii* were identified with stage-specific anti-BAG-1 antibodies (anti-BAG1-7E5; kindly supplied by Dr. Wolfgang Bohne - Institut für Medizinische Mikrobiologie, Universität Göttingen, Germany) as previously described [31]. Initially, the cultures were fixed for 20 min at 4°C on days 1 to 4 with 4% PFA in PBS, washed three times for 10 min in PBS, and then incubated for 30 min in 50 mM ammonium chloride to block free aldehyde radicals. Next, the cells were permeabilized for 20 min in a PBS solution containing 0.05% Triton X-100 (Roche, Rio de Janeiro, RJ, Brazil) and 4% BSA (Sigma-Aldrich-St. Louis, MO, United States) to block nonspecific binding. For the indirect immunofluorescence assay, the host cells were incubated for 2 h at 37°C with the primary antibodies anti-SAG-1 (1:200) and anti-BAG (1:500) diluted in PBS/BSA. After incubation, the cells were washed with PBS containing 4% BSA and incubated for 1 h at 37°C with the secondary antibody at a 1:1000 dilution (anti-mouse IgG conjugated with FITC). Controls were performed by omission of the primary antibodies. Afterward, the cultures were washed 3 times for 10 min in PBS and processed for fluorescence microscopy as described above.

*Development of Schizont Stages of* Toxoplasma gondii *in Primary Cell Culture of Feline… DOI: http://dx.doi.org/10.5772/intechopen.105957*

#### **2.5 Ultrastructural analysis**

FIECs infected or not with bradyzoite forms of *T. gondii* were washed 3 times for 10 min with PBS and fixed for 1 h at 4°C in 2.5% glutaraldehyde diluted in a 0.1 M sodium cacodylate buffer containing 3.5% sucrose and 2.5 mM CaCl2 (pH 7.2). After fixation, the cells were washed in the same buffer and then post-fixed for 30 min at room temperature in 1% osmium tetroxide diluted in a 0.1 M Na-cacodylate buffer. For transmission electron microscopy analysis, the cells were washed in the same buffer, scraped from the plastic dish at 4°C, and centrifuged. Then, the cells were dehydrated in a graded acetone series and embedded in an epoxy resin (PolyBed 812). Thin sections were stained with uranyl acetate and lead citrate and then examined under a transmission electron microscope (Jeol JEM1011). For scanning electron microscopy, the FIECs were fixed for 30 min at room temperature with 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2) and post-fixed for 30 min at room temperature with a solution of 1% OsO4 containing 2.5 mM CaCl2 in the same buffer. The cells were dehydrated in an ascending acetone series and dried by the critical point method with CO2 (CPD 030, Balzers, Switzerland). The samples were mounted on aluminum stubs, coated with a 20 nm layer of gold (Cressington Sputter Coater 108), and examined under a scanning electron microscope (Jeol JSM 6390LV) at the Rudolf Barth Electron Microscopy Platform at Oswaldo Cruz Institute.
