Photo-oxidation Using MiniSOG with EM Preparation of Transfected Culture Cells
Thomas J. Deerinck, Mark H. Ellisman, Mason Mackey, Xiaokun Shu, Varda Lev-Ram, Yingchuan Qi, Ericka B. Ramko, Michael W. Davidson, Yishi Jin, Roger Y. Tsien
Abstract
Abstract taken from Plos Biology Journal: A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms
Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce “miniSOG” (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.
Steps
Cells are grown on glass bottom MatTek culture dishes. (P35G-0-14-C, MatTek Corp)
Transfected Cells are fixed with 37oC 2% glutaraldehyde in *0.1M sodium cacodylate buffer pH 7.4 with 2.0 mM CaCl2 for 5 minutes at room temp and then for 55 minutes on ice.
Wash 5X with 0.1M sodium cacodylate buffer pH 7.4 with 2.0 mM CaCl2 for 2 minutes each on ice.
Block with 50 mM glycine, 10 mM KCN, 10 mM aminotriazole and 0.4mM H2O2 in 0.1M sodium cacodylate buffer pH 7.4 with 2.0 mM CaCl2 for 20 minutes on ice. Added H2O2 just before using.
Wash 2X with 0.1M sodium cacodylate buffer pH 7.4 with 2.0 mM CaCl2 for 2 minutes on ice.
Collect confocal fluorescent images. (Excitation beam 488nm)
Add DAB with 0.22um Millex 33mm PES sterile filter. Let sit for 5 minutes.
Photooxidation using GFP filter (Ex:450-490nm, DM:510nm and Em:515nm LP) with intense light from a 150W xenon lamp with a stream of pure oxygen gently bubbling the DAB solution.
Wash 5X with 0.1M sodium cacodylate buffer pH 7.4 with 2.0 mM CaCl2 for 2 minutes on ice.
Post-fix cells with reduced 1-2% osmium tetroxides (0.8% potassium ferrocyanide + 2.0 mM CaCl2) in 0.1M sodium cacodylate buffer pH 7.4 for 30 minutes on ice.
Wash 3X with 0.1M sodium cacodylate buffer pH 7.4 with 2.0 mM CaCl2 for 1 minutes on ice.
Wash 5X with cold DDH2O, dehydrate 20, 30, 70, 90, 100% ethanol on ice one minute each.
Dehydrate with 100% dry ethanol 3X 1 minute each at room temperature.
Infiltrated with (1:1 ratio) 100% dry ethanol and Durcupan epoxy resin for 30 minutes. with lid on and place on rocker. Make sure both components are mixed completely where the mixture is homogenous.
Add 100% Durcupan epoxy resin overnight.
Next day, 3X Durcupan epoxy resin for one hour each and then place in vacuum oven at 60Co for 48 hours.
