Renlam m 1 resin

Fly heads were separated from the body, dissected into two halves and incubated in fixative solution (4% paraformaldehyde, 2.5% glutaraldehyde in 1 x PBS, pH 7.4) for 1 h at room temperature. The semi-heads were washed 3 times with 0.1 M sodium cacodylate buffer (pH 7.4) for 10 min and postfixed in 2% OsO₄ in 0.1 M cacodylate buffer (pH 7.4) for 1 h. After 3 washes in 0.1 M sodium cacodylate buffer for 10 min the semi-heads were dehydrated through a graded series of ethanol from 30% to 100%. The dehydrated semi-heads were incubated in 100% propylene oxide twice for 10 min each, and then transferred to 50% propylene oxide: 50% Renlam M-1 resin (Serva Electrophoresis, Heidelberg, Germany) and incubated overnight. After that the semi-heads were incubated in 100% Renlam® M-1 resin overnight, embedded in 100% Renlam® M-1 resin and polymerized at 60°C for two days. Ultrathin sections (60–70 nm) were obtained using a Reichert Ultracut E microtome (Leica). Sections were counterstained with heavy metal staining (2% uranyl acetate in 50% ethanol; aq. 2% lead citrate). Ultrathin sections were analyzed in a Tecnai 12 BioTwin transmission electron microscope (FEI, Eindhoven, The Netherlands. Images were obtained with a charge-coupled device SIS Megaview3 SCCD camera (Surface Imaging Systems, Herzogenrath, Germany). Image contrast was adjusted with Adobe Photoshop CS using different tools.