Jem 1400 transmission electron microscope

JEOL JEM-1400 Transmission Electron Microscope with a Gatan OneView® camera was used to image the grids at 120 V. For colocalisation analysis in the human brain, four nuclei per grid, of medium to large size (> 50% of X8000 magnification view), were randomly selected and imaged at X15000-X20000 magnification. Four grids were taken from each case, accounting for one repeat for the two double immunolabelling cases. In all cases, randomised selection was undertaken by identifying nuclei at low magnification (X5000), then imaging at higher magnification. All images were analysed using Image J. For colocalization analysis on brain sections, each observed 15 nm gold particle, signifying a Tau 1 antigen, was checked for colocalisation with 5 nm gold particles, signifying TIP5 antigens. Our definition of colocalisation is; when the number of one antigen (TIP5 particles) within a 5 0 nm radius of the second antigen (Tau 1) is greater than zero (n > 0). Gold particles were included in our analysis if; Tau 1 particles measured between 11≤x≤19 nm and TIP5 particles measured between 1≤x≤9 nm. The method of colocalisation analysis was roughly based on the cross-K function; we used the number of gold particles of the first type at distances shorter than a given distance from a typical particle of the second type divided by the area of the 50 nm inclusion circle [29 ].

Ultrastructural alterations were analyzed by using TEM thin sectioning technique as reported earlier with minor modifications.^{67 (link)} In brief, cells were fixed with 4% paraformaldehyde (PFA) and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for 4 h at 4 °C. Samples were washed in 0.1 M phosphate buffer, post-fixed in 2% OsO₄ at RT and encapsulated in agarose. This was followed by dehydration in ascending grades of ethanol, infiltration and embedding in Spurr Resin and polymerization at 60 °C for 24 h. Ultrathin sections (50–70 nm) were obtained using an ultramicrotome (Leica Ultracut UCT, Leica Microsystems GmbH, Wetzlar, Germany) and picked up onto 200 mesh copper grids. The sections were double stained with uranyl acetate and lead citrate and observed under a Jeol JEM 1400 Transmission Electron Microscope equipped with Gatan ES500 Erlangshen and Gatan Orius SC200B CCD cameras at 80kV (Gatan Inc., Pleasanton, CA, USA). At least 300 cells were analyzed from three independent experiments.