Low molecular weight tannic acid

The fixed kidney samples were cut into 250-μm-thick slices with a DTK-1000 Microslicer (Dosaka EM, Kyoto, Japan), and the slices were processed largely in accordance with a combinatorial heavy metal staining protocol which has been released on the website of the National Center for Microscopy and Imaging Research (La Jolla, CA) (http://ncmir.ucsd.edu/sbfsem-protocol.pdf). This protocol was designed to enhance signal for backscatter electron imaging of epoxy-resin-embedded mammalian tissue at low accelerating voltages. In brief, the tissue slices were successively immersed in 1% osmium tetroxide containing 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 1 hr on ice, 1% low molecular weight tannic acid (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M cacodylate buffer for 4 hr at RT, 2% aqueous osmium tetroxide for 30 min at RT, 1% aqueous uranyl acetate overnight at RT, and Walton's lead aspartate solution36 (link) for 30 min at 60°C. The slices were then dehydrated with a graded series of ethanol, and were embedded in epoxy resin, Oken Epok 812 (Okenshoji, Tokyo, Japan).

Fixed specimens were processed using a combinatorial heavy metal staining protocol for enhancing the signal for the backscatter electron imaging of epoxy-resin-embedded biological samples at low accelerating voltages. In brief, the specimens were successively immersed in 1% osmium tetroxide which contained 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 1 h on ice, 1% low molecular weight tannic acid (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M cacodylate buffer for 4 h at 25 °C, 2% aqueous osmium tetroxide for 30 min at 25 °C, and 1% aqueous uranyl acetate overnight at 25 °C. Subsequently, samples were dehydrated with a graded series of ethanol and embedded in epoxy resin, Oken Epok 812 (Okenshoji, Tokyo, Japan). For the detailed protocol of sample preparation, see Miyaki et al. (2020a (link), b ).