Evo 40 vp

Sample processing was carried out using glass coverslips pre-coated with 1 mg/ml poly-L-lysine. Parasites were fixed for 1 h in 2.5% glutaraldehyde diluted in cacodylate buffer [0.1 M (pH 7.2)]. Cells were then adhered to coverslips, post-fixed for 1 h with 1% osmium tetroxide diluted in cacodylate buffer, and dehydrated in a graded alcohol series (50%, 70%, 90%, and two exchanges of 100% ethanol for 10 min each step). Samples were critical-point dried in a Leica EM CPD030 apparatus (Leica, Wetzlar, Germany). Specimens were coated with platinum in a Leica EM SCD050 before visualization using a Zeiss EVO 40 VP scanning electron microscope.

For SEM, sample processing was performed using 13-mm diameter round glass coverslips, pre-coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA), as previously described. Each coverslip containing the TGCs was fixed for 1 h in 2.5% glutaraldehyde, diluted in cacodylate buffer (0.1 M (pH 7.2)), washed three times with cacodylate buffer 0.1 M, and then post-fixed in the dark for 1 h with 2% osmium tetroxide (OsO₄) diluted in a cacodylate buffer 0.1 M, washed three times with cacodylate buffer 0.1 M, and dehydrated in a graded alcohol series (30%, 50%, 70%, 90%, and three exchanges of 100% ethanol, with each step taking a total of 15 min). The samples were critical-point dried (CPD 030 Bal-tec, Balzers, Liechtenstein), mounted on specific supports, and metalized with gold using a specific metallizer (EM SCD 050, Leica microsystems, Wetzlar, Germany) with a thickness of 18 nm. The visualization was further performed in a Zeiss EVO 40 VP scanning electron microscope [32 (link)].

Parasite processing was carried out using glass coverslips pre-coated with 1 mg/ml poly-L-lysine. Cells were fixed for 1 h in 2.5% glutaraldehyde diluted in cacodylate buffer [0.1 M (pH 7.2)] and then were adhered to coverslips, post-fixed for 1 h with 1% osmium tetroxide diluted in cacodylate buffer. Samples were dehydrated in a graded alcohol series (50%, 70%, 90%, and two exchanges of 100% ethanol for 10 min each step) and then were critical-point dried in a Leica EM CPD030 apparatus (Leica, Wetzlar, Germany). Specimens were coated with platinum in a Leica EM SCD050 before visualization using a Zeiss EVO 40 VP scanning electron microscope. Measurements of cells lengths were made using the program AxioVision4 and were based upon the SEM images. Statistics were calculated using the Wilcoxon-Mann-Whitney test in GraphPad Prism 6 software (GraphPad Software). P values less than 0.05 were considered statistically significant.