Immunogold Labeling of AQP4 and α1-syn

Immunogold labeling was performed as previously described [37 (link)]. Briefly, sections were rinsed in Tris-Buffered saline with Triton X-100 (TBST; 0.05% Tris-HCl, 0.9% NaCl and 0.1% Triton X-100), followed by the incubation with 2% (w/v) human serum albumin (HSA) at room temperature (RT) for 10 min. The sections were incubated with primary antibody overnight, followed by incubation with secondary goat anti-rabbit IgG antibody conjugated to 15 nm colloidal gold particle (1:20 dilution; Abcam; Cat#: ab27236; RRID:AB_954457) for 2 h. The sections were contrasted with 2% uranyl acetate and 0.3% lead citrate for 90 s each and examined using Tecnai 12 transmission electron microscope (FEI, Hillsboro, OR) at 80 kV.
Primary antibodies were: i) affinity-purified rabbit polyclonal antibody against AQP4 (1:400 dilution; Sigma Aldrich; Cat# A5971; RRID:AB_258270); ii) affinity-purified rabbit polyclonal antibody against α1-syn (SYN259; 1:100 dilution) which has previously been validated [38 (link)].