Copper 400 mesh grids

Aggregates were imaged according to a standard protocol [90] (link), [91] . Copper 400 mesh grids (Agar Scientific, Stansted, UK) were coated with Formvar and carbon film. Aggregate solutions were diluted 50-fold in eppendorf tubes, and 5 µl aliquots were placed on the grids. After 60 s, 10 µl of distilled water was added and then excess water was removed. Then, 10 µl of 2% uranyl acetate (Agar Scientific) was placed on the grid and left for 30 s. Finally, two 10 µl drops of distilled water were added and again excess water removed. The grid was then left to dry. Images were collected using transmission electron microscopy (Technai 20, FEI) operating at an acceleration voltage of 120 kV and magnifications typically around ×26,000. It is worth to note that for high hierarchical assembly as amyloid aggregates the staining can be highly heterogeneous, mainly due to the compactness of the structures and salt exclusion. As a consequence, differences in the staining efficiency between different morphologies of aggregates can occur as also evidenced in Figure 8.

To investigate fibrils formed by sole addition of FN075, an aSN stock was mixed with and then applied to a sample grid without further treatment. Moreover, fibril samples obtained both with and without FN075 after fibrillation induced using a platereader were also investigated.
Grids were prepared as described by Smith and co-workers30 (link). Briefly, 6 μL of the sample were loaded onto copper 400 mesh grids (Agar Scientific, Stansted, UK) coated with Formvar and carbon film. After 60 s, 10 μL of distilled water was added and excess water was removed. Subsequently, 10 μL of 2% (w/v) uranyl acetate (Agar Scientific) was placed on the grid and left for 30 s. Finally, 2 × 10 μL distilled water were added and again excess water was removed. The grid was then left to dry. Images were acquired at Core Facility for Integrated Microscopy (CFIM), at the Department of Health and Medical Sciences UCPH. Images were collected using a CM100 transmission electron microscope operating at an acceleration voltage of 80 kV.

TEM was used to visualize the inhibitory activity of aloin by imaging the fibrils present in the samples after 20 hours of fibrillation assay. Three samples were analyzed, namely a sample containing only 1 mg/mL (172 μM) insulin, a sample containing insulin with 400 μM fresh aloin added and a sample containing a two weeks old 400 μM aloin solution. The imaging was performed on a Philips CM 100 Transmission Electron Microscope using a standard protocol [21 (link);22 ]. After 20 hours of fibrillation assay, performed as described above, the samples were diluted 50-fold and 3.5 μl aliquots were placed on Copper 400 mesh grids (Agar Scientific, Stansted, UK) coated with Formvar and carbon film. The grid was left for 60 seconds after which 10 μl of distilled water was added and the excess water was removed. Then, 10 μl of 2% uranyl acetate (Agar Scientific) was added and the grid left for 30 seconds. Last, two 10 μl drops of distilled water were placed on the grid, the excess water was removed and the grid was left to dry. [22 ].