For the past few weeks, I’ve been spending a lot of time performing immunostaining experiments. Immunostaining allows us to determine whether a specific protein is expressed in each cell. It can be performed in tissue samples or in cells grown in vitro. I do a lot of in vitro cell culture, so I performed immunostaining on those samples.
The basic idea behind immunostaining is to use an antibody that detects your protein of interest, which can then be bound by another antibody, allowing detection. For this to work, the primary antibody (which binds the original protein) must be specific to that protein only, so that it does not bind sites in cells that do not express the protein. The secondary antibody (which binds the primary antibody) must also be specific to the primary antibody and must be detectable. We use fluorescent secondary antibodies and image our slides under a fluorescent microscope.
I’ve attached an example of what immunostaining looks like. In this image, the red staining corresponds to Tuj1, a marker of mature neurons. The blue staining corresponds to DAPI, which marks the nucleus of each cell.