Three-dimensional immunogold labeling of nuclear matrix proteins in permeabilized cells.
A preembedment labeling procedure is described for the three-dimensional (3D) labeling of nuclear matrix proteins in permeabilized cells. The procedure is based on the use of ultra-small (1 nm) gold particles as a marker system. This marker penetrates the nucleus more efficiently than the conventionally used 5-10 nm colloidal gold probes. Dehydration is performed by freeze-substitution to preserve the ultrastructure of the cell as optimally as possible. During freeze-substitution the samples are stained by uranyl ions to stain the cellular material throughout the resin section. The 3D gold-labeled and uranyl-stained specimen is embedded in Epon resin and semi-thin (0.2-0.5 microns) sections are made for stereo electron microscopy. The applicability of this method is illustrated by the localization of nuclear matrix-associated nuclear bodies in permeabilized interphase and mitotic HeLa cells.
de Graaf
A
A
Dept. Molecular Cell Biology, University of Utrecht, NL.
Humbel
B M
BM
Stuurman
N
N
van Bergen en Henegouwen
P M
PM
Verkleij
A J
AJ
D002463:Cell Membrane Permeability / Q000187:drug effects; D004853:Epoxy Resins; D017110:Freeze Substitution; D006046:Gold; D006367:HeLa Cells / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron / Q000379:methods*; D015530:Nuclear Matrix / Q000737:chemistry / Q000648:ultrastructure*; D009687:Nuclear Proteins / Q000032:analysis*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology
D004853:Epoxy Resins; D009687:Nuclear Proteins; D011092:Polyethylene Glycols; C004875:EPON; D006046:Gold; D017830:Octoxynol