Immunofluorescence: Definition, Procedure & Applications

Instructor: Amanda Robb
In this lesson we'll be looking at the basic procedure for immunofluorescence. We'll see how to use this technique in the lab and what applications it has in science and medicine.

What Is Immunofluorescence?

Imagine hopping on your bike to go to a friend's house after dinner. In the winter, it gets dark pretty early and you have dark jeans on and a black jacket. How will you ensure that cars and other cyclists see you on the road? Many cyclists attach multiple lights on their bodies and bikes while they cycle at night. A front and rear light help, and some even go the extra mile to add brightly colored lights in their spokes, so their wheels light up at night.

It turns out that the use of brightly colored lights works well not only for bikes, but also in molecular biology laboratories.

Proteins are the working components of cells. They carry out all cell structures and functions. Often, during a disease like cancer or diabetes, how much of certain proteins are made or how the proteins behave is disregulated. Scientists need to track these changes to understand what is causing a disease. But proteins are beyond microscopic - how can we find them in the cell?

Like our cyclist, one answer is immunofluorescence. In this procedure, proteins are tagged with a fluorescent antibody that glows when put under the right wavelength of light. This allows scientists to see proteins that would otherwise be invisible, even under a high powered microscope!

Procedure

1. Fix

First, the cells scientists want to look at need to be frozen in time using a fixative. Cells can be fixed in their growing dishes, but more often they are plated onto microscope slides for easy viewing. Fixatives are chemicals that kill cells and preserve them perfectly. Usually scientists use 4% formaldehyde diluted in a buffer called PBS for this step, similar to what morticians use to preserve human bodies. After the slides are fixed for about 5 minutes, the slides are washed with PBS, usually three times.

2. Permeabilization

Often, the proteins scientists want to look at are inside the cells, but cells are protected by a cell membrane. In order for the immunofluorescent antibodies to access the proteins, scientists must break up the cell membrane in a step called permeabilization. This is usually done using a detergent called Triton-X 100 diluted in PBS to about 0.1 - 0.25%. After permeabilization, the slides are washed with PBS again.

3. Blocking

Antibodies are sticky and sometimes will bind to proteins they are not a match for during staining. This creates background noise and doesn't help scientists locate the actual protein of interest. To stop this non-specific binding, scientists use blocking. During the blocking stage, the cells are incubated with a blocking buffer that contains protein that will bind to non-specific sites on the cells, blocking them from coming in contact with the antibody. Scientists often use bovine serum albumin (BSA) diluted to about 1% in PBS as a blocking buffer between 30 to 60 minutes. After blocking, the slides are washed with PBS.

4. Primary Antibody

Next, it's time to apply the primary antibody. The primary antibody is a perfect match for the protein scientists are looking for. The primary antibody is diluted in PBS according to the manufacturer's instructions and then applied to cells. Scientists might incubate the cells with the primary antibody for one hour at room temperature, or they can let it sit overnight in the cold. It depends on the experiment, the primary antibody and the protein target. After incubation, the slides are washed again with PBS.

5. Secondary Antibody

Now, it's time for the lights! Although sometimes in direct immunofluorescence the primary antibody contains the fluorophore, most of the time there is a secondary antibody applied to amplify the signal and increase sensitivity.

Usually indirect immunofluorescence is used to increase the detection of the protein
immunofluorescence protocol

The secondary antibody is a match for the primary antibody and contains an immunofluorescent tag. Different secondary antibodies attach to different primary antibodies and can have different color fluorophores, so a sample could be stained for more than one protein at a time. The secondary antibody is also diluted in PBS and incubated with the sample, usually for about 1 hour in the dark so the fluorophore is not photobleached by light.

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