Immunofluorescence Method

The purpose of immunofluorescence is to detect the location and relative abundance of any protein for which you have an antibody. Once you have antibodies to your favorite protein, you can use them to indicate where the protein is located. In this example, we will use antibodies for the calcium ATPase, or pump, that is located in the endoplasmic reticulum (ER) of very cell. The antibody used here only recognized the chicken calcium ATPase but immunofluorescence can be used on any protein.

The key to this entire process is the ability to visualize the antibody when looking through a microscope. Since antibodies are smaller than calcium ATPases, you cannot see the antibody directly. Therefore, you have to use a fluorescent dye that is covalently attached to the antibody. When a light illuminates the fluorescent dye, it absorbs the light an emits a different color light which is visible to the investigator and can be photographed.

Figure 1. In most immunofluorescence experiments, two antibodies are employed. The first one, called the primary antibody, is typically generated in a mouse and binds to your favorite protein, which in this case is the chicken calcium ATPase (shown as a series undulating striped line that zigzags through teh ER membrane 10 times). The secondary antibody was purchased from a company that sells antibodies that bind to mouse antibodies and have a fluorescent dye covalently attached to it. As illustrated here, the secondary antibodies can bind to multiple sites on the primary antibody and thus produce a brighter signal since more dyes are brought to a single location.

The first step is to choose your cells of interest. In this case, we will look at a chicken fibroblast, or skin cell. It was grown in tissue culture and so it appears as an isolated cell witn no visible neighbors.

The cell was fixed with formaldehyde to retain the shape and location of all cellular proteins. The cell was treated with a mild detergent to disolve small holes in the membranes so the antibodies could have access to the cytoplasm. Because the calcium ATPase is located in the ER, the antibodies must have access to the cytoplasm or they could not bind to the target protein.

Figure 2. This immunofluorescence micrograph shows the ER being labeled with a monoclonal antibody against the the chicken calcium ATPase. This chicken cell was fixed, permeablilized, and processed for immunofluorescence. White indicates the location of the fluorescent antibody and thus the calcium ATPase to which the antibody was bound. Immunofluorescence photomicrograph by A. Malcolm Campbell.

Using immunofluorescence, investigators can see when, where and how much of their favorite protein is expressed in any cell or tissue. On this web page, you can see other examples where the same antibody was used to labled chicken calcium ATPase in other chicken tissues.


List of Methods

© Copyright 2001 Department of Biology, Davidson College, Davidson, NC 28036
Send comments, questions, and suggestions to: