Figure 2.

Acetylcholine binding on E. histolytica membrane. (A) Trophozoites treated with 1 µM ACh for 1 h were fixed and permeabilized. For immunodetection, anti-acetylcholine-FITC (green) antibody (1:800 dilution) and Hoechst 33342 (blue) for nuclear staining were used. Images were obtained by confocal microscopy at X20. (B) Cellular location of ACh and L220 (as a surface marker) on E. histolytica trophozoites membrane by using anti-acetylcholine-FITC (green) antibody (1:800 dilution), and an anti-L220 (red) antibody (1:500 dilution). Additionally, α-bungarotoxin-Alexa Fluor 647 (1:100) was also used. Nuclei were counterstained with Hoechst 33342 (blue). Representative images of the confocal microscopy analysis at X63. (C) Colocalization between ACh and L220 quantified and compared using Pearson’s and Mander’s correlation coefficients (considering as significant R coefficients values above 0.6 threshold). Data correspond to the mean ± SEM of five independent experiments (n = 5). The statistical analysis was performed with the one-way ANOVA and Tukey posttest method, where the values of ***p < 0.001 were considered significant.