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Immunofluorescence is a common laboratory technique used to examine individual proteins that are in a preserved cell. It can determine both the location and relative quantity of a specific protein. Antibodies are used that specifically recognize and bind to the protein of interest. These antibodies contain a fluorescent tag that absorbs light and emits it at a different wavelength. The fluorescence is then analyzed under a fluorescent microscope and the resulting signal corresponds to the protein's location. Immunofluorescence was first used more than fifty years ago.

General protocol

Immunofluorescence can be very finicky because the ideal protocol depends on the protein that is analyzed, the type of cell line, and the antibody that is used. A lot of fine-tuning generally needs to be done before the signal is optimal. You may notice the similarities between this protocol and the protocol for a technique called Western blotting. This technique uses antibodies to analyze a specific protein present on a membrane instead of in fixed cells.

For clarification, let’s say we’re going to look at nucleolin, a protein present in the nucleus.

  1. The cells that will be analyzed are first spun onto glass microscope slides. A special device called a cytospin, similar to a centrifuge, is used to spin the cells onto the slides. The cells then undergo a process called fixation, where they are placed in certain chemicals such as alcohol or formaldehyde. This essentially freezes the cells and their proteins in place on the slide and helps to preserve the in vivo morphology. It also helps to make the proteins inside the cell accessible to the antibody. Various fixative chemicals are commonly used depending on the cell, protein, and antibody used in the experiment. (See fixation for a more detailed explanation).
  2. The cells are then blocked by placing the slides in a solution containing milk proteins or the protein BSA. These proteins bind to and block nonspecific sites on the cells where the antibody might bind. This helps to minimize background signals.
  3. The slides are then placed in a solution containing the primary antibody. In this case, the primary antibody binds directly to an epitope sequence on nucleolin. Primary antibodies come in two types: monoclonal and polyclonal. Both types are used in immunofluorescence, but monoclonal antibodies generally are more specific and give a stronger fluorescent signal.
  4. After exposure to the primary antibody, the slides are gently washed several times with a mild buffer to remove excess, unbound primary antibody.
  5. The slides are then placed in a solution that contains the secondary antibody. This antibody binds to the primary antibody that is already bound to nucleolin. It does this by recognizing the specific animal used to create the primary antibody. For example, if we used a nucleolin antibody that was created in a rabbit we would used an anti-rabbit secondary antibody. The secondary has a fluorescent tag at the other end that is responsible for emitting the signal. There are many different kinds of fluorescent tags. Typical tags include:
    • Fluorescein or FITC – emits green light when excited with blue
    • Rhodamine or RITC – emits red light when excited with green-yellow
    • Cy5 - emits far red light when excited with red
  6. The slides are washed again with the buffer to remove excess secondary antibody. A coverslip (thin glass square) is placed over the cells and often sealed with clear nail polish to preserve the samples. The slides are then examined under a fluorescent microscope where the fluorescent tag is excited with the proper wavelength and emits a fluorescent signal. This signal will fade if the sample is continually excited, so it is important to minimize exposure to the wavelength. Digital photographs of the fluorescence are generally taken and kept as a record of the signal.

Additional analysis using immunofluorescence

Costaining: Costaining is a method often performed to verify the location of the protein in the cell. For example, since we are looking at nucleolin, a protein that is present in the nucleus, a dye is often used simultaneously that detects the location of the nucleus. The most common nuclear dye is Hoechst, which binds to the DNA present in the nucleus. Hoechst is excited by an UV light and produces a blue fluorescent signal that represents the nucleus. This signal is then aligned with the fluorescence from the nucleolin antibody tag. If the two signals overlap it's generally safe to conclude that nucleolin is in the nucleus. Different stains or antibodies can be used to locate other regions and organelles in the cell. However, keep in mind that immunofluorescence is a two dimensional representation of a three dimensional fluorescent cell. There is a slight possibility that the overlap is because the signal is directly above or below the plane of the other signal, and the two are not actually in the same location.

Colocalization: Immunofluorescence can also be used to determine if two or more proteins share the same location in a cell. Two primary antibodies that recognize the two specific proteins are added simultaneously to the sample. Two secondary antibodies with different fluorescent tags are then added. The sample is then analyzed under the microscope to determine if the two fluorescent signals overlap. If the two fluorescent signals are in the same location then the proteins are located in the same cellular region. This is an indirect way of determining if two proteins interact; if they are located in the same region there is a chance they might bind each other.

Sources: Personal experience and Current Protocols in Cell Biology, Volume 1

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