Epon araldite

Cells were visualised on a ELYRA PS.1 (Zeiss) attached to a LSM 710 inverted microscope (Zeiss) with a 100x oil objective (Zeiss) and a DAPI/GFP/mRFP/Alexa 633 fluorescence filter set. Optical sections (84 nm) were collected through entire cell volumes using 5 grid patterns for structured illumination microscopy. Image stacks were processed using ZEN Black software (Zeiss) and displayed as transparent three-dimensional (3-D) volumetric renderings. Widefield fluorescence microscopy was performed using a FSX100 (Olympus). For transmission electron microscopy, iPS-CMs cultured on Thermanox coverslips (ThermoFisher) were fixed in 2% formaldehyde, 2.5% grade I glutaraldehyde, and 0.03% picric acid in 0.1 M cacodylate buffer, pH 7.4 at 4 °C prior to incubation in 1% osmium tetroxide and 1.5% potassium ferrocyanide dissolved in cacodylate buffer. After contrast staining with 1% uranyl acetate in cacodylate buffer, cells were dehydrated and infiltrated with a 1:1 mixture of Epon-Araldite (Electron Microscopy Sciences) and propylene oxide. Embedding resins were polymerised for 24 to 48 h at 60 °C and 60 nm-thick sections on were placed on copper grids and imaged on a Jeol 1200EX (80 kV).

Persistently infected Efk cells were scraped in phosphate buffered saline and pooled in a 15 ml screw-cap centrifuge tube. The cells were recovered by centrifugation at 300xg for 5 mins and the supernatant was discarded. The cell pellet was resuspended in 10% neutral buffered formalin (10% NBF; Sigma, Cat #HT501128) for virus inactivation. The cells were later processed for electron microscopy as mentioned previously^{25 (link)}. Briefly, cells were treated with osmium tetroxide (1% OsO₄, 0.1 M sodium cacodylate buffer) for one hour at room temperature. Samples were quickly rinsed with water, gradually dehydrated in ethanol and en-bloc stained with uranyl acetate. After rinsing three times (5 min each) in propylene oxide, samples were infiltrated with Epon/Araldite (electron Microscopy Sciences, Cat #50-980-381). Samples were placed in molds and freshly prepared Epon/Araldite was added. The samples were then polymerized at 60 °C for 24–48 h. Sections of 90 nm were cut and observed by transmission electron microscopy (TEM - Hitachi HT 7700, Tokyo, Japan).

Preparation of ultrathin sections of retinal tissue for examination by TEM has been described previously.^{13 (link),14 (link)} Briefly, a 2 × 2 mm piece of retinal tissue was removed 5 mm above the superior margin of the optic disc. Retinal tissue was rinsed in buffer, postfixed in 2% osmium tetroxide and 1.5% potassium ferrocyanide in dH₂O for 2 hours, dehydrated in a graded series of ethanols and embedded in Epon-Araldite (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections (90 nm) of embedded retinal tissue were cut from each block with a diamond knife (Micro Star Technologies, Inc., Huntsville, TX) using an ultramicrotome (Ultracut E 701704; Reichert-Jung, Buffalo, NY). Ultrathin sections were collected on copper grids, and counterstained with 4% methanolic uranyl acetate (Electron Microscopy Sciences). Photoreceptor structure was then examined with a transmission electron microscope (Model 300: Phillips, Eindhoven, The Netherlands).

Mice were perfused with a mix solution of 2% PFA and 2% glutaraldehyde in 0.1 M Cacodylate buffer (Caco). After perfusion, brains were fixed overnight in the same solution at 4°C. The samples were then rinsed three times in 0.1 M Caco buffer. 100 μm thick coronal sections were cut on a vibrating microtome and post fixed with 2% osmium tetroxide in 0.1 M Caco buffer for 2 h at room temperature. Sections were rinsed three times with Caco buffer and then dehydrated through of series of alcohol gradations. The sections were then put into a 1:1 solution of propylene oxide/Epon Araldite (Electron Microscopy Sciences, Hatfield, PA) overnight on an orbital rotator. Sections were then flat embedded with 100% Epon Araldite between 2 sheets of Aclar film and polymerized at 65°C for 48 h. Specific areas of the parietal cortex in these sections were then selected, cut out with a razor blade and glued onto dummy blocks of Epon Araldite. 90 nm ultrathin sections were cut on a Diatome diamond knife, collected on 300 mesh copper grids, and stained with Uranyl Acetate and lead citrate. Grids were viewed on a JEOL JEM1230 transmission electron microscope and images collected with an AMT high-resolution digital camera. Twenty cross-sectional blood vessels were imaged per brain/mouse, n = 3 per group young (4 mo) and aged (18 mo) group.

Samples of tail skin or shaved back skin were immersion-fixed in 1.6% glutaraldehyde in Milloning buffer (0.1M Na₂HPO₄, 0.1M NaHPO₄, pH7.3), for 2 days at 4°C, and subsequently postfixed in 2% osmium for 2h at room temperature. Standard procedures for dehydration and embedding in Epon-Araldite (Electron Microscopy Sciences) were used. Observations were performed using a Tecnai 12 transmission electron microscope. Two mice of each genotype were analysed.

Sections for EM were prepared as previously described^{68 (link)}. Briefly, hemisected eyecups were rinsed in buffer and postfixed in 2% osmium tetroxide and 1.5% potassium ferrocyanide in dH₂O for 2 h, dehydrated in a graded ethanol series, and embedded in Epon-Araldite (Electron Microscopy Sciences, Hatfield, PA). Semi-thin sections (4 μm) were cut and stained with 1% cresyl violet. Ultra-thin sections (90 nm) were cut on an ultramicrotome (Ultracut E 701704, Reichert-Jung, Buffalo, NY) using a diamond knife (Micro Star Technologies, Inc., Huntsville, TX), collected on copper grids, counterstained with 4% methanolic uranyl acetate (Electron Microscopy Sciences, Hatfield, PA), and outer retinal morphology examined using a transmission electron microscope (TEM; Model 300: Phillips, Eindhoven, The Netherlands). Photomicrographs were captured with a digital camera (15-megapixel digital camera, Scientific Instruments and Applications, Duluth, GA) and Maxim DL Version 5 software (Diffraction Limited, Ottawa, Canada).