Cellsens standard program

In order to identify possible changes in leukemic cell morphology, cytospin slides were prepared using a Cytospin centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Leukemic cells (2 × 10⁴ cells/mL) were treated with 100 µM of piperine for 72 h. Subsequently, Quick Panoptic Staining was performed using a commercial hematological kit (Laborclin^® produtos para laboratórios Ltd.a., Pinhais, PR, Brazil). Following the manufacturer’s recommendations, each slide was subjected to a fixative action (0.1% triarylmethane solution) for 1 min. Then, the slides were sequentially immersed in two dye solutions (0.1% xanthene solution and 0.1% thiazine solution) for 1 min each. After the last immersion, the slides were washed in distilled water and air-dried prior to evaluation under a bright field microscopy (OLYMPUS IX81). Images were obtained using the Olympus cellSens Standard program.

Lotus sp. roots were sonicated for 30‍ ‍s for 7 to 10 days after the inoculation with Rhizobium sp. Chiba-1 harboring pFAJDsRed. Roots were examined under Olympus BX53 and Leica M165 FC microscopes. The CellSens Standard program (Olympus) was used to merge images of bright and DsRed fluorescence.

Upon tissue fixation in formalin solution, intestinal samples were dehydrated overnight and embedded in paraffin. Prepared intestinal tissue was cut into 3 µm thick sections and applied to slides for further analysis. All samples were analyzed in a blinded fashion and captured using an Olympus SC 50 camera. Digitization was completed with the cellSens Standard program (Olympus).