*2.6. Immunohistochemistry*

The hearts were completely immersed into five tissue volumes of stabilized 10% neutral formalin solution, which was refreshed after 24 h. After one week, the tissues were placed into cassettes for embedding. The dehydration, clearing, and impregnation processes were carried out in a Leica TP1020 tissue processor (Leica Biosystems, Nussloch, Germany), loading the stations with 10% formaldehyde (2 h), 70% isopropyl alcohol (2 h), 80% isopropyl alcohol (2 h), 90% isopropyl alcohol (2 h), three absolute isopropyl alcohol solutions (2 h each), three xylene solutions (1 h and 30 s for the first solution and 1 h for the others), and two of liquid paraffin (2 h each). For the paraffin embedding of the tissues, a HistoCore Arcadia modular embedding system (Leica Biosystems, Nussloch, Germany) was used. Each block was first put on ice and cut into 4 μm thick sections on a HistoCore MULTICUT microtome with high-profile blades (Leica Biosystems, Nussloch, Germany), then placed in a flotation bath with a temperature of 45 ◦C to deparaffinize the tissue and prepare fragments for deposition onto positively charged slides (Thermo Fisher Scientific 4951PLUS, Cleveland, OH, USA). First, antigen recovery was accomplished by heating at 58 ◦C for 1 h and 30 min. For tissue deparaffinization, the slides were immersed in 3 xylol solutions (at 5-min intervals), in a 50% isopropanol solution (15 immersions), and in tap water (15 immersions). Plates were immersed in a 6% H2O2 solution for 5 min to block endogenous peroxidases, and cleansed. Antigen recovery was carried out by immersing the slides twice into a solution of ethylenediaminetetraacetic acid (EDTA), first at 95 ◦C for 30 min, then at room temperature for 20 min. Each slide was then dipped in a Coplin staining jar containing TBS-T for 10 min, and gently dried with a cotton fiber towel. The tissue area was delimited with a hydrophobic marker (Macrosearch Liquid Repellent). The slides were completely covered with "Ultra V Block" reagent (Ultravision Quanto Detection System HRP DAB kit, Thermo Fisher Scientific, Cleveland, OH, USA) for 5 min, cleared with TBS-T, and dried to remove excess moisture before proceeding with the application of antibodies. For the immunohistochemical technique, 100 μL of the desired antibody was applied to each slide for 1 h at room temperature, and then washed with TBS-T. The antibodies were diluted with Antibody Diluent Ventana® (Roche) as follows: 1:1000 for Anti TNF-α (Abcam Reference ab6671, rabbit polyclonal to TNF-α); 1:250 for Anti IFN-γ (Abcam Reference ab216642, rabbit polyclonal to IFN-γ); 1:200 for Anti-Nitric Oxide Inducible Synthase iNOS (Abcam Reference ab15323, rabbit polyclonal to iNOS); 1:1000 for Anti IL-4 (Abcam Reference ab9811, rabbit polyclonal to IL-4); and 1:250 for IL-10 (Abcam Reference ab217941, and rabbit polyclonal to IL-10). To amplify the primary antibody reaction, a Primary Antibody Amplifier Quanto reagent (Ultravision

Quanto Detection System HRP DAB kit, Thermo Fisher Scientific, Cleveland, OH, USA) was applied, followed by incubation for 10 min and rinsing with TBS-T. For revealing, HRP Polymer Quanto reagent (Ultravision Quanto Detection System HRP DAB kit, Thermo Fisher Scientific, Cleveland, OH, USA) and diaminobenzidine (DAB) (provided in the kit) was applied, following the manufacturer's instructions. Finally, the slides were stained with hematoxylin-eosin. The process was completed with the adhesion of a 24 mm × 60 mm coverslip on each treated specimen. Brain, tonsils, and hearts from rats infected with *T. cruzi* were used in a standardized immunohistochemistry technique. An individual slide was used for each antibody to avoid possible contamination or cross-reaction.

For reading, the slides were scanned on a Ventana DP 200 whole-slide scanner (Roche Diagnostics, Rotkreuz, Switzerland), with the immunoreactivity calculation performed using free QuPath v. 0.2.3 software for digital pathological image analysis [13]. For this purpose, a project and a pixel classifier (px) were created with the following parameters: (i) Moderate resolution (2.00 μm/px); (ii) channel DAB; (iii) Gaussian prefilter; (iv) smoothing sigma 1; and (v) Threshold 0.25 (threshold value, positive results ≥ 0.25), defining the region of interest (ROI) as all cardiac tissue present on the slide. The results were expressed as a global percentage of immunoreactivity of the studied antibody.
