Transmission electron microscopy assay

Transmission electron microscopy (TEM) is used todetect the presence of fibrils. For quality TEM data, the composition of the protein sample solution must not damage the EM grids and excessive salt should be avoided since salt crystals can obscure the identification of fibrils. The grids for amyloid fibril detection can be made of copper or nickel with 200–400 mesh spacing.The grids are first covered in formvar and subsequently carbon coated. Staining is typically performed with a 2% solution of uranyl acetate (UA) in water. Phosphotungstic acid (PTA) at pH 7.0 can also be used for staining fibril samples but significantly improved resolution for fibrillar samples has not been observed with PTA compared to UA. Many things on an EM gridare artefacts that can be mistaken for protein aggregates.For example, blemishes on formvar caused bysome solvents or by a poor formvar coating can appearto be interesting shaped protein aggregates. Folded formvar can look like fibrils but is easily distinguished from authentic fibrils by a dramatic change upon focusing due to the depth of the folded formvar. Denseuranyl acetate crystals are occasionally present and arenot related to the protein composition of the sample. A completely blank grid is observed if the formvar has torn (which occurs if too high a concentration of sampleis used) or if all the protein remained in solution and didnot settle on the grid. The lack of any protein adsorptionto the grid is typically a good indication that there is little to no protein aggregation, amyloid or otherwise.

Procedure for negative staining of amyloid fibrils for TEM:

1. Prepare a solution of 2% uranyl acetate in DDI water. This solution can be stored for several weeks at 4 °C. On the day of sample staining, spin the uranyl acetate solution at 12,000 rpm for 3 min to pellet any unsolubilized uranyl acetate.

2. Place 3μL of protein sample on the grid. After 3 min, apply a torn edge of hardened, ashless filter paper at the edge of the grid to absorb the remaining liquid (this process is known as wicking).

3. Immediately (i.e., do not let the sample dry on the grid), place 3μL of staining solution on the grid, wait 3 min, wick away excess solution, and air dry.

4. The grids can be immediately examined or stored in an EM grid case prior to examination. The grids are imaged using an electron microscope operating at 80 keV. Scan at low magnification (10–12,000X) to get an idea of the overall sample composition and then examine the finer details of the fibrils at higher magnification (25,000X). Amyloid fibrils are typically linear, unbranching, and 5–10 nm in width.