Ethanol hmds

The Johns Hopkins microscopy facility performed scanning electron microscopy. Briefly, tracheas were cut open lengthwise and fixed overnight at 4°C in a buffer of 2.5% glutaraldehyde, 100 mM sodium cacodylate, 3 mM MgCl₂, pH 7.2. Following a rinse in a buffer of 3% sucrose, samples were post-fixed for 1.5 hours on ice in the dark with 2% osmium tetroxide in 100 mM cacodylate buffer containing 3 mM MgCl₂. Samples were rinsed in dH₂O and dehydrated through a graded series of ethanol to 90%. Dehydration was continued through 100% ethanol, then passed through a 1:1 solution of ethanol:HMDS (Hexamethyldisiloxazne Polysciences) followed by pure HMDS. Samples were then placed in a desiccator overnight to dry. Tracheal pieces were attached to aluminum stubs via carbon sticky tabs (Pella), and coated with 40 nm of AuPd with a Denton Vacuum Desk III sputter coater. Stubs were viewed on a Leo 1530 FESEM operating at 1 kV and digital images captured with Smart SEM version 5.