Immunogold Techniques in Electron Microscopy

Abstract

Ever since electron microscopy became an important tool in the scientific research, the focus had been mainly on ultrastructural analysis with little success in the development and application of suitable techniques for the localization of macromolecules in cells. The emergence of immunogold techniques in the 1960s managed to fill this gap in serving this function. The aim of this chapter is to equip researchers, postgraduate students, and technicians with essential knowledge to utilize immunogold techniques for ultrastructural investigations in the life sciences. The principles and factors involved have been highlighted to give researchers a quick review of the techniques before embarking on their ultrastructural localization procedures. The advantages and limitations of the four types of immunogold labeling techniques have been discussed.

Ultrastructure of cell trafficking pathways and coronavirus: how to recognise the wolf amongst the sheep

“…In immunoelectron microscopy experiments, a much lower percentage of glutaraldehyde is usually used to optimise the antigen. Tissue embedded in acrylic resin rather than the standard epoxy resin will result in better antigen preservation, related to low-temperature polymerisation [73]. Ultrastructural in situ hybridisation uses negative sense riboprobes and is best accomplished on tissues in a hydrophilic acrylic resin [72].…”

“…Standard glutaraldehyde and paraformaldehyde fixatives with uranyl acetate and lead citrate staining may be compatible with this technique, although this depends on the antibody. However, osmium tetroxide post‐fixation may interfere [73]. In immunoelectron microscopy experiments, a much lower percentage of glutaraldehyde is usually used to optimise the antigen.…”

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“…The selection of an appropriate antibody in immunolocalization assays is extremely crucial to gain reliable results, in particular, considering that choices have to be made on the basis of availability, quality of antibodies and specificity towards a given protein. The achievement of significant results in immunolocalization investigations deals with the aptitude of the primary antibody to bind specifically proteins through an important biochemical interaction that leads to the formation of the antibody-antigen complex [ 51 ]. The present method, being characterized by the sequential binding of primary, and secondary antibodies, is generally known as indirect method.…”

“…Remarkably, the indirect method in the recent years has gained in popularity compared to the direct IGS procedure that is based on the GNPs directly bound to the primary antibodies. The reason for that may be found in the higher sensitivity of the indirect method as a result of the capacity of secondary antibodies to interact with multiple epitopes located on primary antibodies [ 51 , 52 ]. Because of the sequential steps of incubations with primary and secondary antibodies, it should be noticed that indirect methods may imply the concurrence of cross-reactions with unrelated molecules [ 51 ].…”

“…The reason for that may be found in the higher sensitivity of the indirect method as a result of the capacity of secondary antibodies to interact with multiple epitopes located on primary antibodies [ 51 , 52 ]. Because of the sequential steps of incubations with primary and secondary antibodies, it should be noticed that indirect methods may imply the concurrence of cross-reactions with unrelated molecules [ 51 ]. It follows that suitable and appropriate control samples are of utmost importance.…”