Karnovsky s fixative

Polarized IECs grown on 0.33-cm^{2 (link)} collagen-coated Transwells (Thermo Fisher Scientific) were apically infected with EPEC strains cultured in DMEM. IECs were fixed in Karnovsky’s Fixative (Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C, neutralized with 125 mmol/L glycine in PBS, postfixed in 1% osmium tetroxide, and sequentially dehydrated with 15%, 30%, 50%, 70%, 90%, and 100% ethanol. Samples then were infiltrated with Spurr’s Resin (Electron Microscopy Sciences). Ultrathin sections were contrasted with 2% uranyl acetate, followed by Reynold’s lead citrate, and visualized with an FEI Tecnai Spirit transmission electron microscope (FEI, Hillsboro, OR).

We used SEM to examine spore germination, the growth on leaf surfaces, and entry points into leaves by endophytes. Leaf discs stored in 1% glutaraldehyde were transferred to half-strength Karnovsky’s fixative (Electron Microscopy Sciences, Hatfield, PA, USA) for 4 h and rinsed with 0.1 M of phosphate buffer three times for 20 min each, followed by an ethanol series dehydration (in sequence: 30%, 50%, 70%, 80%, 90%, 95%, and 100%; modified from [23 ]). Each dehydration step was 25 min. Discs then were immersed in 100% ethanol overnight before critical point drying using a Polaron Critical Point Drier (Polaron, Hertfordshire, UK). Leaf discs were mounted on SEM stubs using carbon conductive tabs and then coated with platinum using a Hummer 6 Sputtering coater (Anatech, CA, USA, Hayward, CA, USA). Sample stubs were examined by Hitachi S-4800 Field-Emission (Hitachi, Tarrytown, NY, USA) SEM at the University of Arizona’s University Spectroscopy and Imaging Facilities (USIF). We quickly examined each leaf disc under low magnification and then took several photos at random with a magnification of 300× once patches of conidia or fungal growth were found. At this magnification, the area represented in each photo was approximately 422 µm × 276 µm. The number of conidia per photo was counted for reference.

Mice were anesthetized with ketamine/xylazine (100 mg kg⁻¹ and 20 mg kg⁻¹). Live animals were perfused via the aorta with 10 ml of sodium cacodylate buffer (0.1 M, pH 7.4) followed by 10 ml of 1⁄2 Karnovsky’s fixative in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences). Eyes were enucleated and the anterior segment removed. The eyecups were dehydrated and embedded in tEPON-812 epoxy resin. Semithin Sects. (1 μm) were stained with 1% toluidine blue in 1% sodium tetraborate aqueous solution for light microscopy. Ultrathin Sects. (80 nm) were cut from each sample block using a Leica EM UC7 ultramicrotome (Leica Microsystems) and stained with 2.5% aqueous gadolinium triacetate hydrate and Sato’s lead citrate stains using a modified Hiraoka grid staining system [51 (link)]. Grids were imaged using an FEI Tecnai G2 Spirit transmission electron microscope (FEI Company) at 80 kV interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques) for digital TIFF file image acquisition. TEM imaging of retina samples was assessed, and digital images were captured at 2 k × 2 k pixel, 16-bit resolution.