Em uc6 fc6 cryo microtome

For ultrastructural analysis, the salivary glands were dissected, fixed in 2% formaldehyde/ 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, post-fixed in 1% OsO₄, dehydrated in ethanol and propylenoxide, and embedded in EMbed-812 (EMS, Fort Washington, PA) in flat molds. After polymerization for 60 h at 65°C, 70nm sections were cut on a Leica Ultracut E microtome (Leica, Austria), placed on collodion/carbon-coated grids, and stained with 2% uranyl acetate/lead citrate. Sections were viewed on a Tecnai 12 transmission electron microscope (TEM) (FEI, Hillsboro, OR). For EM immunocytochemistry, samples were prepared according to Tokuyasu (1980) (30 (link)). In brief, the dissected salivary glands were fixed in 4% formaldehyde/0.2% glutaraldehyde in 0.1M PHEM (60 mM PIPES, 25 mM HEPES, 2 mM MgCl₂, 10 mM EGTA, pH 6.9), cryo-protected in 2.3 M sucrose, mounted on aluminum pins, and frozen in liquid nitrogen. Thin frozen sections were then cut on a Leica EM UC6/FC6 cryo-microtome (Leica, Austria), collected on a drop of sucrose/methylcellulose mixture and placed on a formvar-carbon grid. The sections were labeled with primary antibody, and the label was subsequently visualized by colloidal gold conjugated to Protein A. Sections were stained/embedded in 2% methylcellulose/0.2% uranyl acetate and observed under a Tecnai 12 TEM.