Formvar carbon coated 400 mesh copper tem support grids

The α-syn fibrils were immunogold-labeled ‘on grid’ for dityrosine9 (link) using a monoclonal anti-dityrosine antibody (JaICA, cat. no. MDT-020P). The antibody has been fully characterized and shown to be highly specific and does not show any cross-reactivity with other tyrosine derivatives such as nitrotyrosine, chlorotyrosine42 (link). The specificity and the antibody were also checked in our previous work using the identical procedure and IgG concentrations, with an irrelevant antibody to hair cell antigen (MAb10)9 (link).
The antibody will detect any protein containing dityrosine. Briefly, 4 μl aliquots of the α-syn fibrils were pipetted onto Formvar/carbon coated 400 mesh copper TEM support grids (Agar Scientific, Essex, UK), left for 1 min, the excess was removed by filter paper, and then blocked in normal goat serum (1.10 in PBS+) for 15 min. Grids were then incubated with (10 μg/ml IgG) mouse dityrosine monoclonal antibody (JaICA, Shizuoka, Japan) for 2 h at room temperature, rinsed in 3×2 min PBS+, and then immunolabeled in a 10 nm gold particle-conjugated goat anti-mouse IgG secondary probe (GaM10 British BioCell International, Cardiff, UK; 1.10 dilution) for 1 h at room temperature. After 5 × 2 min PBS+ and 5 × 2 min distilled water rinses, the grids were negatively stained as described in negative stain TEM methods below.

Briefly, the immunogold labelling was performed by placing 4 μL of a dGAE filament preparation for 1 min on Formvar/carbon‐coated 400‐mesh copper TEM support grids (Agar Scientific), and then the excess was removed using filter paper. For blocking, grids were incubated with normal goat serum (1 : 10 dilution) for 30 min at room temperature and then incubated with mAb 423 (1 : 20 dilution) for 2 h at room temperature. (mAb 423 was raised against PHFs from AD brains and detects tau protein C‐terminally truncated at Glu‐391 as reported in 19, 26). Grids were washed three times with PBS+ for 2 min each and then incubated with GaM10 secondary antibody (1 : 10 dilution) for 1 h at room temperature. The grids were washed five times with PBS+ for 2 min each followed by five washes with distilled water (0.22‐μm‐filtered) for 2 min each. The filaments were negatively stained with 2 % (w/v) uranyl acetate (0.22 μm filtered) for 1 min. TEM projection images were collected using a JEOL JEM1400‐Plus Transmission Electron Microscope operated at 100 kV equipped with a Gatan OneView camera (4k × 4k). Images were recorded at 25 fps with drift correction using GMS3.