Formvar carbon film coated copper grids

For negative-staining transmission electron microscopy, samples were spread on 300 meshed formvar/carbon film–coated copper grids (Ted Pella, Redding, CA) for 5 min, washed once with distilled water for 1 min, stained with 1% (wt/vol) uranyl acetate for 3 min, and visualized under an Hitachi H-7700 electron microscope (Hitach High-Tech, Japan) operating at 100 kV. Images were acquired using a charge-coupled device camera before being processed further for figures using Adobe Photoshop CC (Adobe System, San Jose, CA).
To quantitate filament width and length, random series of at least five electron micrographs were selected from each in vitro assembly experiment. Widths were measured from 12–15 randomly selected filaments, and quantitative analyses were made using ImageJ software. Because of the aberrant associations of many mutant GFAP filaments, the true length of GFAP filaments was difficult to assess. Therefore, lengths were estimated from representative filaments and expressed as the range of this population. Where the average filament length was especially great, it was then expressed as greater than the length, which could be accurately measured. For some GFAP mutants, short filaments were indicated by an increase in the number of free filament ends seen in electron micrographs.