Quorum q150t

For scanning electron microscopy of platelets, PRP was diluted tenfold with Tyrode's buffer (4 mM HEPES; 135 mM NaCl; 2.7 mM KCl; 2.4 mM MgCl₂; 3.3 mM NaH₂PO₄; 5.6 mM D-glucose; pH 7.4), after which it was fixed with glutaraldehyde at a final concentration of 2.5% and deposited on a polycarbonate filter (0.1 or 0.4 μm pore size) by centrifugation at 500 g, 5 min, with fast acceleration and slow deceleration. The precipitated platelets were washed with phosphate buffer (0.1 M NaH₂PO₄, 0.1 M Na₂HPO₄, pH 7.2), then they were dehydrated with ascending concentrations of ethanol and incubated in hexamethyldisilazane followed by drying. Dehydrated samples were sputter-coated with a thin layer of gold–palladium on Polaron e5100 or Quorum Q150T instruments and used for scanning electron microscopy (Quanta 250 FEG microscope, ThermoFisher Scientific).

Unmated and mated females were fixed for 1 h in 4% FA after the removal of legs and wings, washed 3 × 5 min in PBS (0.85% NaCl, 1.4 mM KH2 PO4, 8 mM Na2 HPO4, pH 7.4) and incubated in 25% sucrose overnight at 4 °C. Flies were briefly washed in PBS before embedding in O.C.T (#4583, Tissue-Tek) and freezing on a LN2 cooled metal block. Using a cryostat (Leica CM1950), 20 µm sections were collected on super plus glass slides (Thermo Scientific) and coated with 1 nm AuPd (Quorum Q 150 T ES, UK).