Visualizing poly(GA) protein fibrils in mouse brain

To examine the filamentous structure of poly(GA) proteins in mouse brain, immunoEM was performed as previously described^{52 (link)}. Thin sections were pretreated with sodium citrate buffer, pH 6 at 90–95 °C for 10 min before immunolabeling. Rabbit polyclonal antibody to poly(GA) (1:150 in PBS) was used as the primary antibody, and goat anti-rabbit IgG conjugated with 18 nm colloidal gold particles (1:20, Jackson ImmunoResearch Laboratories) was used as the secondary antibody. Thin sections stained with uranyl acetate and lead citrate were examined with a Philips 208S electron microscopy (FEI) fitted with a Gatan 831 Orius charge-coupled device (CCD) camera (Gatan). Digital images were processed with Adobe Photoshop CS5 software.

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Zhang Y.J., Gendron T.F., Grima J.C., Sasaguri H., Jansen-West K., Xu Y.F., Katzman R.B., Gass J., Murray M.E., Shinohara M., Lin W.L., Garrett A., Stankowski J.N., Daughrity L., Tong J., Perkerson E.A., Yue M., Chew J., Castanedes-Casey M., Kurti A., Wang Z.S., Liesinger A.M., Baker J.D., Jiang J., Lagier-Tourenne C., Edbauer D., Cleveland D.W., Rademakers R., Boylan K.B., Bu G., Link C.D., Dickey C.A., Rothstein J.D., Dickson D.W., Fryer J.D, & Petrucelli L. (2016). C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins. Nature neuroscience, 19(5), 668-677.

Publication 2016

goat anti-rabbit IgG conjugated with 18 nm colloidal gold particles

Anti igg Antibody Brain Buffer Citrate Colloidal gold Device Electron microscopy Filamentous Goat Mouse Poly Proteins Rabbit Sodium citrate Thin sections Uranyl acetate

Corresponding Organization :

Other organizations : Mayo Clinic in Florida, Jacksonville College, Johns Hopkins Medicine, Johns Hopkins University, USF Health Byrd Alzheimer's Institute, University of South Florida, Harvard University, Massachusetts General Hospital, German Center for Neurodegenerative Diseases, University of Colorado Boulder, Mayo Clinic

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Variable analysis

independent variables

Pretreatment of thin sections with sodium citrate buffer, pH 6 at 90–95 °C for 10 min before immunolabeling

dependent variables

Filamentous structure of poly(GA) proteins in mouse brain

control variables

Rabbit polyclonal antibody to poly(GA) (1:150 in PBS) used as the primary antibody
Goat anti-rabbit IgG conjugated with 18 nm colloidal gold particles (1:20, Jackson ImmunoResearch Laboratories) used as the secondary antibody
Thin sections stained with uranyl acetate and lead citrate

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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