Fig. 3. : effect of MDL28170 and anti-Dm-calpain antibody in the interaction process of Phytomonas serpens with the explanted salivary glands of Oncopeltus fasciatus. (A) Agglutination of parasites with anti-Dm-calpain antibody, which was only achieved up to 1:125 dilution. (B) Differential interference contrast microscopy (DIC) and confocal scanning images showing the double labeling of P. serpens cells for calpain-like proteins (CALPs) cross-reactive to anti-Dm-calpain antibody (green labeling) and DAPI (blue labeling). The labeling in non-permeabilised cells suggests the location of CALPs in the membrane and in the flagellum, and DAPI stained the nucleus (n) and the kinetoplast (k). At right, the overlay of anti-Dm-calpain and DAPI labeling. Bars: 5 µM. (C) Parasites (107 cells) were either pre-treated or not (control) with DMSO (diluent of the drug) or with the ½ x IC50, IC50 or 2 x IC50 values of MDL28170 for 4 h at 28ºC. In parallel, cells were pre-incubated with the anti-Dm-calpain (at 1:250, 1:500 and 1:1000 dilutions) or with a non-immune IgG antibody for 1 h. The parasite viability of the parasites was not affected by the treatments used in this set of experiments. Following interaction for 1 h, the salivary glands were washed and homogenised, and the released trypanosomatids were counted in a Neubauer chamber. The results are shown as the mean ± standard deviation of three independent experiments and the asterisks denote significant differences compared to control (p < 0.05).