Premium
Immunogold labelling in combination with cryoultramicrotomy, freeze‐etching, and label‐fracture
Author(s) -
Boonstra J.,
Bergen en Henegouwen P. M. P.,
Belzen N.,
Rijken P. J.,
Verkleij A. J.
Publication year - 1991
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1991.tb03078.x
Subject(s) - immunogold labelling , labelling , monoclonal antibody , chemistry , antigen retrieval , replica , biophysics , antigenicity , antigen , nanotechnology , materials science , antibody , biology , biochemistry , immunology , art , visual arts
SUMMARY During the past years, the methods of ultrastructural visualization of intracellular and cell‐surface proteins have been improved considerably, mainly as the result of the development of low‐temperature preservation in combination with immunocyto‐chemical labelling procedures using poly‐ or monoclonal antibodies. In this contribution we will discuss the combination of immunogold labelling with cryoultramicrotomy and two replica methods, i.e. freeze‐etching and label‐fracture. The main advantage of cryoultramicrotomy is that it enables post‐sectioning labelling, thus providing complete accessibility of all cellular antigens, located both intracellularly and on the cell surface. Important parameters that influence the labelling (i.e. label‐efficiency), including penetration of the label and antibodies in the section, effects of fixatives on antigenicity, and steric hindrance, will be discussed in detail. The replica methods have the advantage of enabling an analysis of the lateral distribution of antigens located at the cell surface. The label efficiency is of particular importance in these studies and in this context several parameters will be discussed, including accessibility and effect of fixatives.