Lysates contents were decanted for 5 min at room temperature. When specified, 10 μM bafilomycin or 100 μM sodium vanadate were added to the vesicle suspensions for 30 min at room temperature. After decanting, 20 μl cell lysate were applied to Formvar-coated grids and blotted dry with a filter paper. Grids were dried and examined in a JEOL 1200 EX transmission electron microscope operating at 80 kV. X-rays were collected

for 90 s using a Si (Li) detector with Norvar window on a 0–10 keV energy range with a resolution of 10 eV/channel. Semi-quantitative elemental analysis was performed as described (Miranda et al., 2004c). The atomic% was calculated based on the measured weight% values (wt.%/atomic wt.). Larva midguts were dissected and fixed in 4% formaldehyde, 2.5% glutaraldehyde and 0.1 M sodium cacodylate pH 7.2 for 2 h. Cells were washed with Afatinib 0.1 M sodium cacodylate pH 7.2 and post-fixed with 1% OsO4, 0.8% FeCNK, 5 mM CaCl2 for 1 h at dark. Samples were washed in 0.1 M sodium cacodylate pH 7.2, dehydrated in an acetone graded series and embedded in progressive Epon concentrations. Epon-embedded samples were hardened at 60 °C for 72 h, 80 nm ultrathin sections were prepared on an ultramicrotome and mounted

on copper grids. Lead citrate and uranyl acetate were used for post-staining and grids were observed on JEOL 1200EX transmission electron microscope operating Bcl-2 inhibitor at 80 kV. Alternatively, midgut sections were frozen using a high-pressure freezing machine Bal-Tec HPM-010 and 1-hexadecene as cryoagent. Freeze-substitution was performed using 1.45% KF as a calcium-precipitating agent, 3% glutaraldehyde, 1% OsO4 in methanol (Hardt and Plattner,

2000). Samples were kept at −80 °C for 72 h, −20 °C for 6 h, 4 °C for 4 h very and transferred to room temperature. Samples were washed with acetone and embedded in Epon as described above. To better understand the general morphology of the midgut of A. gemmatalis, we prepared histological sections from Historesin embedded samples. No significant morphological differences could be found between anterior and posterior midgut at this level. Anticarsia midgut is divided in three main regions: the endoperitrophic and ectoperitrophic space (EnS and EcS, respectively) and the cellular monolayer ( Fig. 1A), composed of columnar, goblet and regenerative cells ( Fig. 1B). EnS is surrounded by the peritrophic membrane (PM) and defines the inner region of the midgut lumen. This region has been defined as involved with primary digestion ( Terra and Ferreira, 1994), which is corroborated by the observation of undigested food ( Fig. 1A, C, and D). The PM and the cellular monolayer limit the EcS and no food residues could be found. Several vesicles of different sizes and aspects are present dispersed around the EcS and eventually in close proximity to the PM ( Fig. 1C).