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).
Methanolic uranyl acetate
Manufactured by Electron Microscopy Sciences
Methanolic uranyl acetate is a chemical compound used in electron microscopy as a staining agent. It is a solution of uranyl acetate dissolved in methanol. This product is used to enhance the contrast of biological samples during transmission electron microscopy (TEM) imaging.
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4 protocols using methanolic uranyl acetate
1
Retinal Tissue Ultrastructural Analysis
2
Outer Retinal Morphology Examination Protocol
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Sections for EM were prepared as previously described68 (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).
3
Transmission Electron Microscopy of Larvae
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Freshly euthanized larvae were fixed overnight in Karnovsky’s fixative40 , rinsed in 0.1 M sodium cacodylate buffer, post-fixed for one hour in 1% osmium tetroxide, then again rinsed in 0.1 M sodium cacodylate buffer. Larvae were then dehydrated in a graded series of ethanol, infiltrated with and embedded in epoxy resin, and polymerized in a 70 °C oven for 48 hours. To identify areas of interest for TEM analysis, semi-thin sections of 1 µm thickness were cut transversely using an ultramicrotome, stained with toluidine blue and imaged with a Nikon Instruments Eclipse compound light microscope. Ultra-thin sections of 100 nm thickness for TEM analysis were then cut using an ultramicrotome and stained with 33% methanolic uranyl acetate for 15 minutes and lead citrate (Electron Microscopy Sciences, cat # 22410) for 7 minutes. Digital electron micrographs were collected using a JEOL JEM-1230 equipped with a Gatan CCD camera.
4
Transmission Electron Microscopy of Retinal Tissue
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Sections for EM were prepared as previously described 46 (link) . Briefly, hemisected eyecups were rinsed in buffer and postfixed in 2% osmium tetroxide and 1.5% potassium ferrocyanide in dH2O for 2 hr, 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).
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).
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