Megaview g2 digital camera

H661 cells were plated at 1 × 10⁵ cells per well in 6-well plates. DAPT was added as previously described. At the end of a 24 h incubation cells were washed with PBS once, fixed with 2.5 % glutaraldehyde at 4 °C for 2 h and then washed twice with PBS. Cells were dehydrated in a graded series of ethanol (50, 70, 80, 90 and 100 %), transferred in to 100 % acetone for 15 min and embedded in SPURR resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined and photographed with a JEOL 100S transmission electron microscope equipped with an Olympus MegaView G2 digital camera.

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.