Immunogold Labeling of Aquaporin-4 in Tissue

Immunogold labelling of AQP4 was performed on-grid and was based on a previously published method^{49 (link)}. Briefly, matched grids for PTS and control tissue were prepared in triplicate and floated on 50 mM glycine in PBS and blocked with 2% normal goat serum in PBS. Overnight, grids were incubated at room temperature with an AQP4 antibody (A5971, Merck, Australia) at a 1:200 dilution in blocking solution. Secondary blocking occurred prior to room temperature incubation with a 1:20 dilution of anti-rabbit IgG conjugated to 10 nm gold nanoparticles (G-7402, Sigma, Australia) in blocking solution. Sections on-grid were contrast enhanced by incubation with uranyl acetate replacement stain (2 h) and Reynold’s lead citrate (150 s) and allowed to dry overnight. Grids were deidentified for blinded analysis and imaged using a Megaview G2 digital camera (Olympus SIS, Münster, Germany) attached to a Philips CM10 TEM.