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.
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.
Free full text: Click here
