Fig. 3. Promastigote orientation shortly after internalization. Macrophages untreated (white bars) or treated with IFN- (hatched and black bars) were infected with L. amazonensis metacyclic promastigotes. They were fixed 30 minutes (white and hatched bars) or 60 minutes (black bars) after the addition of the parasites and permeabilized before staining with an anti-leishmanolysin or anti-Leishmania immune serum and adequate fluorochrome conjugates. Cell preparations were then examined by fluorescence microscopy. Three groups of intracellular parasites were distinguished according to their orientation as shown in the schemes below the histograms. Results are expressed as the means+s.d. of two (white bars) or three experiments (hatched bars) or are from a single experiment (black bars). Percentages were determined after counting 703, 2200 and 712 parasites, respectively.