Immunogold Labeling of Resin-Embedded Electron Microscopical Sections - Plant Cell Morphogenesis: Methods and Protocols - page 177

Chapter 15

Immunogold Labeling of Resin-Embedded Electron
Microscopical Sections

Ilse Foissner and Margit Hoeftberger

Abstract

Knowledge about the spatio-temporal distribution patterns of proteins and other molecules of the cell is
essential for understanding their function. A widely used technique is immunolabeling which uses specifi c
antibodies to reveal the distribution of molecular components at various structural levels. Electron micros-
copy offers the highest resolution of morphological techniques and is thus an indispensable tool for the
analysis of molecule distribution patterns at the subcellular level. In this chapter we describe a routine method
for labeling ultrathin sections of resin-embedded material with antibodies conjugated to colloidal gold.

Key words Colloidal gold, Immunolabeling, Transmission electron microscopy, Resin-embedded
sections, Antibodies

1 Introduction

1.1 General

Immuno techniques use antibodies to detect, characterize, and
localize specifi c antigens in cell extracts (e.g., dot and Western
blots, immunoprecipitation) and in situ inside tissues and cells
(immunolabeling or immunocytochemistry). To study their distri-
bution antibodies must be conjugated to markers which differ
according to the method used for observation [ 1 ]. Fluorescent
dyes and enzymes which produce colored products with chromo-
genic substances are suitable for fl uorescence and bright fi eld
microscopy. For electron microscopy (EM) electron dense gold
particles are commonly used [ 2 ]. These gold particles can be
adsorbed to macromolecules by complex electrochemical interac-
tions and are available in distinct sizes. The colloidal gold method
is applicable to most of the electron microscopical systems and
techniques (transmission EM, scanning EM; ultrathin sections and
freeze fracture). After increasing the size of gold particles by silver
enhancement, the distribution of gold-conjugated probes can also
be visualized in the bright fi eld microscope.