Genasis software

Slides submitted to classical cytogenetic procedures (conventional, G- and C-banding) were analyzed using a Leica DM-100 optical microscope. Camera control and image acquisition was performed using GenAsis software (Applied Spectral Imaging, Carlsbad, CA, USA). FISH experiments were analyzed using a Zeiss Imager II microscope (ZEISS, Jena, Germany), and images were captured and processed using a CCD camera and Axiovision 4.8 software (ZEISS, Jena, Germany).

GTW-banded karyotype analysis was performed as a standard clinical protocol and described according to the International System for Human Cytogenetic Nomenclature (Arsham and Shaffer 2017 ). FISH techniques were performed as standard clinical protocol. Briefly, for the 13;17 translocation, interphase nuclei were probed using a FLT3 (13q12.2) break-apart probe (Agilent), comprised of flanking probes, one of which covers ∼300 kb of FLT3, at exons 20–24, and another one that covers ∼300 kb of FLT3, at exons 1–9. Samples were analyzed under an Olympus BX53 photoscope, and representative photographs were taken using GenASIs software from Applied Spectral Imaging. A minimum of 100 nuclei were scored and cases were considered positive when >15% of cells displayed split signals.

Experiments were approved by the ethics committee (CEUA—Universidade Federal do Pará) under no. 170/2013. The sample included two females of Amazona aestiva (AAE) and one Pyrrhura frontalis (PFR) male and female (Table S1). Tissue cell cultures from skin biopsies or feather pulp, performed according to Sasaki et al. [18 (link)], with modifications, were used to make chromosome preparations. For harvesting, we used a protocol including colcemid treatment (0.05% for 1 h), followed by incubation with hypotonic solution (KCl 0.075 M) and fixation. For the determination of diploid number and chromosome morphology, slides were stained with Giemsa (5% in phosphate buffer pH 6.8) and analyzed with 100× objective. A minimum of 20 metaphases per individual were photographed and karyotyped using Genasis software (Applied Spectral Imaging, Carlsbad, CA, USA).

Chromosomal analysis was carried out by the standard G-banding protocol (10) with minor modifications. The culture was set up in a 15-ml centrifuge tube with 500-µl whole blood and 5 ml PB - Peripheral blood max media (Gibco USA). Phytohemagglutinin (Phytohemagglutinin Form M: Gibco USA), 300 µl was added to the culture as a mitogen and incubated at 37°C in 5% CO ${}_2$ (Panasonic) for 68 hr.
Colcemid (0.08 µg/ml) (Gibco, USA) 45 µl was added to arrest the cells at the metaphase stage at the 68^th hr.
Next, 9 ml of 0.075 M KCl (Merck, India) was added and incubated at 37°C for 13 min. Then, the cells were fixed by using a 3:1 ratio of methanol (LOBA Chemie, India) and acetic acid (Merck, India) (Carnoy's fixative). The cell pellet was dropped on slides and aged overnight at 60°C hot air oven (Ascension Innovation) overnight. G-banding using Trypsin Giemsa was done by treating with 0.05% trypsin and staining with 4% Giemsa stain.
Twenty well-spread metaphases with excellent band resolution were selected for analysis under oil immersion (100 $\times$ ) using a bright field microscope (BX53, Olympus, Japan).
Karyotypes were reported using the GENASIS Software (Version 7.2, Applied Spectral Imaging, Israel). Identified chromosomal abnormalities were designated as per the International System for Human Cytogenetic Nomenclature 2013 guidelines (11).

Fresh fertilized eggs (PintoBar, Lda, Portugal) were incubated at 37 °C in a humid environment. After 6 days of embryonic development, a window was opened into the eggshell under aseptic conditions. On day 10, Kyse-410 cells suspension in growth factor-reduced Matrigel (BD Biosciences) were seeded on CAM. Then, on day 13, a treated group, randomly selected, received IOX1 50 μM whereas a control group received only 1% DMSO in complete RPMI-1640. After 24 h, CAM was irradiated with 2 Gy. Lastly, on day 17, tumors were dissected and included in a paraffin block. Microtumor images were obtained on day 13 (0 h of treatments) and at day 17 (72 h of treatment). Relative perimeter in in ovo was assessed using CellSens software (version V0116, Olympus). Ex ovo pictures were obtained for blood vessels counting using Image J software.
Immunostaining of microtumors’ sections was evaluated through a quantitative method using GenASIS software (Applied Spectral Imaging, ASI). Staining’s evaluation was performed as described in the tissue immunoexpression subsection.

Metaphase-chromosome spreads were prepared from 80% confluent cultures according to standard procedures. Actively dividing cells were treated with 10 ng/ml colcemid (Gibco KaryoMAX Colcemid solution in PBS, Thermo Fisher Scientific) for 16 hr (overnight) at 37°C. Cells were combined in 0,56% KCl for 20 min at 37°C and were fixed with methanol/acetic acid (3:1 v/v). Chromosome analysis was carried out by applying Q-banding by fluorescence using quinacrine (QFQ), according to routine procedures, following the guidelines of the International System for Chromosome Nomenclature 2009 (ISCN 2009) (Shaffer et al., 2009 (link)). Microscope observation was performed using the Fluorescence microscope Olympus BX63, fully equipped with quinacrine mustard filter and CCD camera and the acquisition and analysis of ‘GenASIs’ Software, version 8.1.0.47741 (Applied Spectral Imaging). On average, 25 metaphases were evaluated. No gross chromosomal alterations were observed by Q-banding (Figure 7—figure supplement 2B).