Transmission Electron Microscopy Specimen Processing

Human specimens were fixed in 1/2 strength Karnovsky’s fixative and processed for TEM using standard procedures, as described previously^{70 (link), 71 (link)}. Briefly, specimens were rinsed in cacodylate buffer, post-fixed in 2% osmium tetroxide, en bloc stained with 2% uranyl acetate, dehydrated with alcohol to propelene oxide, and infiltrated with tEPON-812 epoxy resin (Tousimis, Rockville, MD) utilizing an automated EMS Lynx 2 EM tissue processor (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections (80 nm) were cut from the epoxy block using a Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL) and a diamond knife. Sections were imaged using an FEI Tecnai G2 Spirit transmission electron microscope (FEI, Hillsboro, Oregon) at 80 kV interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques, Woburn, MA).

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Benischke A.S., Vasanth S., Miyai T., Katikireddy K.R., White T., Chen Y., Halilovic A., Price M., Price F J.r., Liton P.B, & Jurkunas U.V. (2017). Activation of mitophagy leads to decline in Mfn2 and loss of mitochondrial mass in Fuchs endothelial corneal dystrophy. Scientific Reports, 7, 6656.

Publication 2017

tEPON-812 epoxy resin Leica EM UC7 ultramicrotome Tecnai G2 Spirit transmission electron microscope XR41 digital CCD camera

Buffalo Buffer Cacodylate Camera Dehydrated alcohol Diamond Electron microscopy Epoxy Fixative Human Lynx Microscopy Osmium tetroxide Oxide Tissue Transmission electron microscope Ultramicrotome Uranyl acetate

Corresponding Organization :

Other organizations : Massachusetts Eye and Ear Infirmary, Harvard University, Smith-Kettlewell Eye Research Institute, Price Vision Group, Duke University

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Variable analysis

independent variables

Fixation in 1/2 strength Karnovsky's fixative
Processing for TEM using standard procedures

dependent variables

Ultrastructural characteristics of the human specimens observed under the transmission electron microscope

control variables

Rinsing in cacodylate buffer
Post-fixation in 2% osmium tetroxide
En bloc staining with 2% uranyl acetate
Dehydration with alcohol to propelene oxide
Infiltration with tEPON-812 epoxy resin using an automated EMS Lynx 2 EM tissue processor
Cutting of ultrathin sections (80 nm) using a Leica EM UC7 ultramicrotome and a diamond knife
Imaging using an FEI Tecnai G2 Spirit transmission electron microscope at 80 kV interfaced with an AMT XR41 digital CCD camera

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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