Ultracut uct 7 link ultramicrotome

hTCEpi cells were seeded onto 35 mm glass-bottom dishes (MatTek Corporation, Ashland, MA, USA) containing KGM and allowed to adhere overnight. Cells were treated for 24 h in KBM with or without rhIGFBP-3. Following treatment, 2.5% glutaraldehyde/0.1 M cacodylate buffer pH 7.4 was added to the cells for 15 min at room temperature to allow for fixation. Next, cells were washed three times for five minutes with 0.1 M sodium cacodylate buffer. Following washing, cells were post-fixed in 1% osmium tetroxide and 0.8% K₃[Fe(CN₆)] in 0.1 M sodium cacodylate buffer for 1 h at room temperature and then rinsed with water. Cells were next stained overnight en bloc with 2% aqueous uranyl acetate and then washed with water. Cells were then dehydrated through exposure to increasing concentrations of ethanol and infiltrated with embed-812 resin and polymerized at 60 °C overnight. Once embedded, cell blocks were sectioned on a Leica Ultracut UCT [7 (link)] ultramicrotome (Leica Microsystems, Heidelberg, Germany) using a diamond knife (Diatome, Hatfield, PA, USA) and positioned on copper grids. Finally, samples on copper grids were post-stained with 2% uranyl acetate in water and lead citrate. Imaging was performed using a JEOL 1400 Plus (JEOL) equipped with a LaB₆ source using a voltage of 120 kV.