4.5. Anatomical Damage

To observe the damage inside the tissues, samples were processed according to previous protocols [66], in which leaves or leaflets were fixed in a FAA solution for 24 h, then rinsed with distilled water. They were then dehydrated in a series of tertiary butyl alcohol-ethanol-water mixes (Table S2). Subsequently, they were infiltrated and embedded in histological paraffin at 58 °C; paraffin blocks were obtained, and samples were oriented to obtain cross sections.

The 15-μm thick histological sections were cut in an American Optical 820 rotation microtome (US), stained with safranine-fast green, and finally mounted on synthetic resin. Observations were made using an Axioskope photomicroscope (Carl Zeiss, Oberkochen, Germany), and photographs were taken from the middle vein, intercostal area, and margin from each species and treatment, using a video camera Exwave HAD (Sony, Tokyo, Japan). Finally, the photographs were edited in the GIMP 2.10.6 software, to remove some stains in the preparation-ns.