4.3 Immunolabelling

Immunolabelling is a technique used to detect specific proteins or structures in cells or tissues by using antibodies — molecules that naturally recognise and bind to particular targets. A so-called primary antibody (yellow Y-shapes in Figure 12a) binds to a target, for example the protein actin. Primary antibodies are raised in different animals. The so-called primary antibody is either directly attached to a fluorescent dye or detected by a second fluorescently labeled antibody (green and red Y-shapes in Figure 12a). When viewed under a fluorescence microscope, the dye lights up, allowing researchers to see where the protein is located in the sample. A common variation of this technique is double immunolabelling, where two different proteins are labeled at the same time using antibodies tagged with two distinct fluorescent dyes, or by using two primary antibodies that were raised in different animal species. These primary antibodies are recognised specifically by different secondary antibodies, labelled with different fluorophores.

Double-immunolabelling allows scientists to observe the spatial relationship or co-localisation of different proteins within the same cell or tissue (Figure 12). Immunolabelling can only be performed in fixed and permeabilised cells, because the large antibody molecules would otherwise not be able to enter the cells. By using labels other than fluorophores, immunolabelling can also be visualised with light and electron microscopy.