**2.3 Obtention of sera from murine model of acute and chronic Chagas' disease**

The murine model of acute and chronic Chagas' disease is already established in the Chagas Disease laboratory of the Instituto Dante Pazzanese. For the proposed study, a total of 8 male mice, Black-C57, 20 days old, were inoculated intraperitoneally with 1x104 parasites per milliliter (mL) in 0.9% saline, pH 6.0, and three animals were used as negative controls.

After infection, 200 to 400 uL of blood samples were collected from the mice on days 7, 15 (acute phase of infection), 30, 45, and 60 (chronic phase of infection) after infection, through submandibular vein puncture, and centrifuged at 1200 x g for 10 minutes at 25°C, to obtain the serum, which was stored at −20°C until analysis. On day 60, the mice were euthanized in a chamber with cotton soaked in Sevoflurane (C4H3F7O), following cervical dislocation to ensure death. After euthanasia, a necropsy of brain tissue, liver, spleen, kidneys, and heart was performed. The heart was washed with saline and cardiac tissue and saline lavage were stored at −20°C. The euthanasia method was performed according to the recommendations of the Brazilian guide to good practices for the euthanasia of animals established by the consultants of the Ethics, Bioethics and Animal Welfare Committee of the Federal Council of Veterinary Medicine (CEBEA/CFMV). All handling of the animals was carried out in accordance with the Brazilian guideline for the care and use of animals for scientific and educational purposes (DBCA), of the National Council for the Control of Animal Experimentation (CONCEA) (https://www.sbcal.org.br/download/download?ID\_ DOWNLOAD=58). The protocol was approved by the Animal Research Ethics Committee of the Instituto Dante Pazzanese (Registration number 020/2019).

*T. cruzi* infection was confirmed by reading slides containing blood smears and by polymerase chain reaction (PCR) with the oligonucleotides Tc121 (5′ AAATAATGTACGGGKGAGATGCATGA 3′) and Tc122 (5′ GGTTCGATTGGGGTTGGTGTAATATA 3′), which amplifies a 330 bp fragment corresponding to *T. cruzi* kDNA [45, 46]. The PCR reaction was performed with the enzyme Platinum Taq DNA polymerase (ThermoFischer Scientific), according to the supplier's instructions. The cycles used for amplification were 3 min 94°C/40x 94°C 1 min; 62°C 1 min; 72°C 1 min/7 min 72°C. PCR products were analyzed on 2% agarose gel stained with ethidium bromide.
