Immunogold labelling in combination with cryoultramicrotomy, freeze-etching, and label-fracture.
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 immunocytochemical 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.
Boonstra
J
J
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
van Bergen en Henegouwen
P M
PM
van Belzen
N
N
Rijken
P J
PJ
Verkleij
A J
AJ
D000818:Animals; D005613:Freeze Etching; D005614:Freeze Fracturing; D007150:Immunohistochemistry; D008867:Microtomy