Axiophot microscope

The hearts and liver tissues were fixed in formalin, dried through increasing concentrations of alcohol, and embedded in paraffin wax. Two micrometer-thick paraffin slice were cut from paraffin-tissue pieces. The tissue areas were deparaffinized by immersing in xylene also, were rehydrated by decreasing concentration of alcohol. The sections were then stained either for Masson’s trichrome or with hematoxylin and eosin (H and E) and then rinsed with water. Each slide was dried out thoroughly with alcohol. Photomicrographs were obtained using Zeiss Axiophot microscope. For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), the tissue sections were treated with proteinase K (2 μg/mL) for 15 min and were then washed in PBS twice. The section were then flooded with 0.1% sodium citrate (with 0.1% Triton X-100), absorbed blocking cradle and washed twice with PBS. The sections were subsequently kept in TUNEL reagent (Roche Applied Science, Indianapolis, IN, USA) for 60 min at room temperature. 4,6-diamidino-2-phenylindole (DAPI) reagent was used to stain the nucleus. The nucleus was fluoresced by blue light at 454 nm and the TUNEL-positive cells fluoresced in brilliant green at 460 nm. Photomicrographs were obtained using Zeiss Axiophot microscope.

The lizards were restrained manually, and their blood was collected by cardiac puncture into heparinized (100 U mL⁻¹) 1 mL syringes. The blood smears and the impression smears (imprints) of the lungs, brain, heart, kidney, liver, bone marrow and spleen were air-dried, fixed with absolute methanol and stained with 10% Giemsa Methylene Blue Eosin Merck^® diluted in distilled water (pH 7.0) for 50 min, according to Eisen and Schall (2000 (link)), to allow analysis of the different parasite forms and stages. The samples were examined, and the infected cells were photographed with a Zeiss Axiophot microscope using a 100× immersion objective. Prevalence was estimated as the proportion of infected hosts, expressed as a percentage. The intensity of parasitaemia in blood monocytes was graded according to Silva et al. (2004 (link)) as negative, low-level infected, medium-level infected and highly infected.
Measurements of the length, width and area of the gamonts and host cells (infected and uninfected) were performed. Morphometric data are presented in micrometres (μm), and for each metric, the averages, ranges and standard deviations were also calculated. Measurements of cells were carried out using a 100× oil immersion objective on a Zeiss Axiophot microscope, calibrated with a stage micrometre. Unstained samples were analysed by differential interference contrast (DIC) microscopy.