Tif annotation editor

Samples were fixed with 0.1 M sodium cacodylate (Sigma), 2% gluteraldehyde (Electron Microscopy Sciences), 3% PFA (Electron Microscopy Sciences), 5% sucrose buffer (Sigma) and 1% osmium tetroxide (pH 7.4) (Electron Microscopy Sciences). The samples were then dried in increasing concentrations of high-grade ethanol, followed by critical point drying using Autosamdri 815 critical point dryer and sputter coated using Cressington 208HR sputter coating with Au or Pt/Pd. Imaging was done on a Jeol 5600LV SEM, Zeiss EVO SEM or Zeiss FESEM Ultra55 microscope. For each image the total number of cancer cells, cancer cells with nanotubes, cancer cells without nanotubes, total number of nanotubes, total number of EPI–EPI membrane nanobridges, EPI–ENDO nanobridges, number of cells forming EPI–EPI nanobridges, EPI–ENDO nanobridges and number of cells positive for both EPI–EPI and EPI–ENDO nanobridges were counted. Length and width of the nanobridges were measured using the CarlZeiss TIF annotation editor. Width was measured at three different positions across the length of the nanobridges and the average width was calculated for the comparison of length and width of the nanobridges.