Tannic acid-mediated osmium impregnation after freeze-substitution: a strategy to enhance membrane contrast for electron tomography.
In this technical note we report a tannic acid-mediated osmium impregnation method that, applied after freeze-substitution, increases membrane contrast in cells for transmission electron microscopy and tomography studies. The general staining that is achieved allows visualization of organelles, plasma membrane and associated specializations (e.g. caveolae) in non-post-stained plastic sections by conventional transmission electron microscopy. In combination with electron tomography it results in membranes with a proper contrast and equal staining pattern through the depth of the tomograms. The protocol that we contribute can serve as starting point for those willing to improve the membrane contrast of their specimens or to make 3D studies on the architecture of membranous compartments by electron tomography.
Jiménez
Nuria
N
Department of Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. N.JimenezGil@uu.nl
Vocking
Karin
K
van Donselaar
Elly G
EG
Humbel
Bruno M
BM
Post
Jan A
JA
Verkleij
Arie J
AJ
D002021:Buffers; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure*; D055032:Electron Microscope Tomography / Q000379:methods*; D042783:Endothelial Cells / Q000648:ultrastructure; D005057:Eukaryotic Cells / Q000648:ultrastructure; D005404:Fixatives / Q000737:chemistry; D017110:Freeze Substitution / Q000379:methods*; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D009993:Osmium Tetroxide / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*; D013634:Tannins / Q000737:chemistry*
D002021:Buffers; D005404:Fixatives; D013634:Tannins; D009993:Osmium Tetroxide