Panoptic stain

Intracellular parasite loads were analyzed as previously described [19 (link)]. Briefly, cells were infected and treated with nucleotides and were fixed onto slides, stained using a panoptic stain (Laborclin®, PR, Brazil), and counted using a Primo Star light microscope (Zeiss, Germany), with a 40x objective (100x for representative pictures). Images were acquired using a Bx51 camera (Olympus, Tokyo Japan) operated using the Cell^F software. We calculated the “infection index,” representing the overall infection load, based on the count of about 100 cells in a total of five fields to obtain the number of infected macrophages and the average number of parasites per macrophage. Individual amastigotes were clearly visible in the cytoplasm of infected macrophages. The results were expressed as the infection index II = (%infected macrophages) × (amastigotes/infected macrophage)/100.

Mice were euthanized with a lethal dose of ketamine/xylazine (240 and 30 mg/kg, respectively, i.p.), blood was collected from the abdominal vena cava for antibodies levels (serum) evaluation, and BAL was performed. For that, the mice trachea was exposed, a 1.7-mm catheter was inserted, and two aliquots of 1 mL of PBS 1X were flushed three times into the bronchoalveolar compartment to recover the leukocytes and fungi in the airways of mice. The cells were centrifuged and resuspended in 100 µL of PBS 1X and used to total (Neubauer chamber) and differential cell counts and fungi counts. Cytocentrifuge preparations (Shandon III) stained with Panoptic stain (Laborclin) were used for differential counts of leukocytes, based on morphological criteria.
Pulmonary tissue was extracted after perfusion of 5 mL of cold sterile PBS 1X in order to remove circulating blood. The right lung of mice was collected for indirect quantification of neutrophils (myeloperoxidase assay - MPO), macrophages (N-acetyl-beta-D-glucosaminidase - NAG), and eosinophils (eosinophil peroxidase - EPO) recruited into the tissue. The left lobe of the lungs was fixed in buffered formalin 10% for 48 hours, for further histological examination.