Detecting proteins with antibodies

Immunohistochemistry and immunocytochemistry are widely used techniques in which a protein of interest is specifically recognized and tagged by an antibody (immunolabeled).

During immunolabeling, in order to allow the binding of the antibody to the protein of interest, cells have to be treated with chemicals, which damage cell structures. For this reason, first of all cells are treated with chemicals preserving cell structures from the subsequent treatments. One of the most common fixative is paraformaldehyde that, by creating links between proteins inside the cell, fixes (1.) its morphology.

After fixation, cells are treated with detergents, that create pores in the plasma membrane (2.): cells become permeable.

After “blocking” unspecific sites (to mask parts of other proteins that might interact with low specificity with the antibody), cells are incubated with a solution containing the primary antibody, which means an antibody specifically recognizing the protein of interest. The antibody enters the cells through the pores created with the permeabilization, and binds the protein (3.). In the scheme, the protein is expressed on the external nuclear membrane.

After washing away the excess of primary antibody that did not bind the protein, cells are incubated with a solution containing a secondary antibody. Secondary antibody recognizes and binds the primary antibody (4.). The secondary antibody is linked to a fluorescent probe.

Now cells can be mounted on a microscope glass and images can be acquired (5.). In the figure (5.), the cell (a retinal neuron) has been immunolabeled with an antibody recognizing a cytoplasmic protein, expressed both in the cell body and in the axon, and detected thanks to a secondary antibody conjugated to a green fluorescent probe.

The same process can be performed in tissue slices, allowing to visualize the expression and localization of a protein in a cell or in a tissue.

However, what if we want to see this protein moving inside the cell following a stimulus? Sure, we could fix the cell before and after the stimulus and compare the protein distribution, but what if in that short period the protein moves and then comes back to the original situation? We would see no changes by comparing the two images, while a lot happened.

For this reason, live imaging has become more and more popular. Although immunocytochemistry is undoubtedly useful, only following a protein behaviour in real time will allow to precisely determine what is going on. As the long treatments pre-antibody incubation kill the cells, antibody-based techniques are not suitable for this kind of analysis, and molecular biology has supported scientists in finding a way around the problem.