Anti nedd4 antibody

Postmortem human SNpc paraffin-embedded sections from sporadic PD patients and control individuals were obtained from the Neurological Tissue Bank (Biobank-HC-IDIBAPS) and stained as previously reported [6 (link)]. Sections were dewaxed with xylene and rehydrated by incubation in ethanol series (100%, 95%, 70% and 50%). Antigen retrieval was performed with Tris-EDTA buffer (10 mM Tris-base pH = 9, 1 mM EDTA, 0.05% Tween 20) in a vegetable steamer at 100°C for 20 minutes. Then, slides were blocked in superblock-PBS for 2 hours at room temperature and were incubated with avidin/biotin solution (Vector Laboratories, Burlingame, CA). Sections were incubated O/N at 4°C with anti-NEDD4 antibody (Abcam), washed with TBS-0.025% Triton X-100 and incubated for 15 minutes with 0.3% H₂O₂ in TBS. Secondary antibody incubation was performed for 2 hours at room temperature with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories). Finally, slides were incubated with ABC Peroxidase Standard Staining Kit (Thermo Fisher Scientific) for 30 minutes and with ImmPACT SG Peroxidase Substrate (Vector Laboratories) for 20 minutes. Sections were washed, dehydrated by incubation in ethanol series (50%, 70%, 95% and 100%) and xylene, mounted with DPX mountant (Sigma-Aldrich) and examined under the microscope.