0.1 m sodium cacodylate buffer

Salmonella-infected HeLa cells grown on Thermanox® coverslips (Ted Pella, Inc., Redding, CA) were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA). All subsequent processing steps were carried out in a Pelco Biowave laboratory microwave system (Ted Pella, Inc.) at 250 W. Following fixation, the monolayer was rinsed with buffer and post fixed with 1% osmium tetroxide reduced with 0.8% potassium ferrocyanide in 0.1 M sodium cacodylate buffer under 20 in. Hg vacuum. After rinsing in 0.1 M sodium cacodylate buffer, the monolayer was treated with 1% aqueous tannic acid and en bloc stained using 1% aqueous uranyl acetate under vacuum. The cells were then rinsed with distilled water and dehydrated in a gradient ethanol series. The monolayer was infiltrated under vacuum with 1∶1 (ethanol: Spurr’s resin) and 100% resin. The cells were later embedded in resin and sectioned on a UC6 ultramicrotome (Leica Microsystems, Vienna, Austria). Sections were collected on a 200 mesh copper grid, stained for contrast using 4% uranyl acetate and lead citrate prior to imaging on a Tecnai BioTwin Spirit TEM (FEI, Hillsboro, OR). Digital images were acquired with a Hamamatsu Orca digital camera system (AMT, Danvers, MA.).

To image hair-cell bundles, zebrafish larvae were exposed to strong water wave stimulus, then anesthetized in 0.12 % tricaine in E3 and immediately fixed in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences) supplemented with 2 mM CaCl₂. Larvae were shipped overnight in fixative, then most of the fixative (~90–95%) was removed, replaced with distilled water, and samples were stored at 4 C. Next, larvae were washed in distilled water (Gibco), dehydrated with an ascending series of ethanol, critical point dried from liquid CO₂ (Tousimis Aurosamdri 815), mounted on adhesive carbon tabs (Ted Pella), sputter coated with 5 nm of platinum (Leica EM ACE600), and imaged on Hitachi S-4700 scanning electron microscope. Kinocilia diameter measurements were performed using ImageJ.

For visualization of live and fragmented bacteria on FcMBL beads, bacteria were captured with 128 nm FcMBL beads (Ademtech, France), spun down onto 13=-mm coverslips and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences, USA) for 1 hour. Cover slips were incubated in 1% osmium tetroxide in 0.1 M sodium cacodylate (Electron Microscopy Sciences, USA) for 1 hour. Ascending grades of ethanol dehydrated the sample before being chemically dried with hexamethydisilazane (Electron Microscopy Sciences, USA). Samples were then placed in a desiccator overnight. Dried samples were mounted on aluminum stubs, sputter-coated with a thin layer of gold particles, and imaged using a Zeiss Supra55VP microscope.

Mouse optic nerve samples were collected and fixed with half strength Karnovsky’s fixative (2% formaldehyde + 2.5% glutaraldehyde, in 0.1 M sodium cacodylate buffer, pH 7.4 (Electron Microscopy Sciences, Hatfield, Pennsylvania) for a minimum of 48 hours. After fixation, samples were rinsed with 0.1M sodium cacodylate buffer, post-fixed with 2% osmium tetroxide in 0.1M sodium cacodylate buffer, then dehydrated with graded ethyl alcohol solutions, transitioned with propylene oxide and resin infiltrated in tEPON-812 epoxy resin (Tousimis, Rockville, Maryland) utilizing an automated EMS Lynx 2 EM tissue processor (Electron Microscopy Sciences, Hatfield, Pennsylvania). Processed tissues were oriented in tEPON-812 epoxy resin and polymerized in silicone molds using an oven set for 60°C for 48 hours. Semi-thin cross-sections were cut at 1-micron with a Histo diamond knife (Diatome, Hatfield, Pennsylvania) on a Leica UC-7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL) and collected on slides then dried on a slide warmer. The slides were stained with 2% aqueous paraphenylenediamine (MP Biomedicals LLC, Solon, Ohio) solution for 45 minutes at room temperature, rinsed in tap and deionized water solutions, air-dried, then mounting medium and a glass coverslip was applied over the sections for light microscopic analysis of myelinated axon analysis.