Immunofluorescence (IF) Protocol and FAQs

Immunofluorescence (IF) is an experimental method that combines immunological principles and fluorescence techniques. It is widely used in biological research, especially in cell biology, molecular biology and pathology. Immunofluorescence detects and localizes the distribution of specific proteins or other molecules in cells or tissues by binding specific antibodies to the target antigen and by fluorescently labeled secondary antibodies or directly labeled antibodies.

The Principle of Immunofluorescence

Immunofluorescence uses the binding between antibodies and their specific antigens to identify target molecules. The antibody is highly specific to the target antigen and can accurately bind to the target protein or other molecules. Antibodies are usually attached with fluorescent tags (such as FITC, Cy3, etc.). These fluorescent dyes can emit visible light under excitation light irradiation. Through different fluorescent dyes, multiple target molecules can be detected in the same sample. The samples were observed by fluorescence microscope after fluorescence labeling. Fluorescence microscopy can excite fluorescent dyes at specific wavelengths to emit fluorescence at specific wavelengths, thereby showing the position of the target molecule in the cell or tissue.

The Steps of Immunofluorescence

Sample Preparation

Sample preparation is a very important step in immunofluorescence experiments. The purpose is to ensure that the target molecule remains stable and the antibody can effectively enter the cell or tissue for labeling. Cells or tissue sections are fixed with fixatives (such as formaldehyde, glacial acetic acid, or ethanol) to maintain the integrity of the cell structure while preventing degradation of the target protein. The commonly used fixative is 4 % paraformaldehyde (PFA), which can be fixed at room temperature and usually takes 10-30 minutes. In order to allow the antibody to penetrate the cell membrane or tissue, surfactants (such as Triton X-100, Saponin or Tween-20) are usually used for osmotic treatment. The penetrant destroys the integrity of the cell membrane, allowing the antibody to enter the cell. In order to reduce non-specific binding, cells or tissue sections were blocked with blocking solution (usually a buffer containing BSA (bovine serum albumin) or normal animal serum). This step usually lasts 30 minutes to 1 hour.

Antibody Incubation

The antibody incubation step is a key step in immunofluorescence, involving the binding of specific antibodies to target antigens. Specific primary antibodies (i.e. antibodies that bind to the target antigen) were added and allowed to bind to the target molecule in the sample. Primary antibodies are usually incubated overnight at 4℃ or incubated at room temperature for 1-2 hours. Secondary antibody incubation (if using indirect immunofluorescence) : If indirect immunofluorescence is used, after the primary antibody binds to the target antigen, a fluorescently labeled secondary antibody is added. Secondary antibodies are antibodies against primary antibodies, usually extracted from different species, which can enhance the signal.

Washing

The washing step is to remove the antibody that is not bound to the antigen and reduce the interference of the background signal. Wash the sample several times with PBS (phosphate buffered saline) or TBS (Tris buffered saline), usually 5-10 minutes each time, to remove unbound antibodies and other impurities.

Fluorescence Staining

In this step, the target molecule in the cell or tissue will be shown by fluorescently labeled antibodies.

If the indirect immunofluorescence method is used, the secondary antibody will label the fluorescent dye. Commonly used fluorescent dyes include FITC (fluorescent isothiocyanate green), Cy3 (red), Alexa Fluor series dyes, etc.

Sample Seal

In order to facilitate observation and prevent the sample from being dried or damaged under a microscope, it is usually necessary to use a sealing liquid to seal the film. The sample was sealed using a sealing solution containing an anti-fluorescence attenuator (such as a sealing solution containing DAPI). The sealing solution can not only protect the sample from the influence of the external environment, but also enhance the fluorescence signal.

Fluorescence Microscope Observation

Finally, the samples were observed using a fluorescence microscope. The fluorescence microscope can excite the fluorescent dye in the sample and detect the distribution of the target molecule. Fluorescent dyes emit visible light of a specific wavelength through an excitation light source (usually ultraviolet light or blue light). Different fluorescent dyes have different excitation and emission wavelengths, and multiple labeling targets can be observed through different filters.

1 Sample preparation : fixed, infiltration, closed.
2 Antibody incubation : primary antibodies (or primary and secondary antibodies) were added.
3 Washing : Remove unbound antibodies.
4 Fluorescent staining : labeled fluorescent dye.
5 Sealing : use the sealing liquid to seal the film.
6 Fluorescence microscope observation : Observe and record the fluorescence signal.

Figure 1. Direct immunofluorescence. (Reference source: An introduction to Performing Immunofluorescence Staining.）

Figure 2. Indirect immunofluorescence. (Reference source: An introduction to Performing Immunofluorescence Staining.）

Figure 3. Amplification of signal with polyclonal secondary antibodies labeled with a fluorophore. (Reference source: An introduction to Performing Immunofluorescence Staining.）

Figure 4. Amplification of signal by a secondary antibody conjugated with multiple fluorophore protein complexes. (Reference source: An introduction to Performing Immunofluorescence Staining.）

Figure 5. Amplification of signal with polyclonal secondary antibodies conjugated with multiple fluorophore–protein complexes. (Reference source: An introduction to Performing Immunofluorescence Staining.）

Advantages of Immunofluorescence

High sensitivity : Fluorescence labeling has high sensitivity and can detect low-expressed proteins or molecules.

Spatial localization : It can clearly show the specific location and distribution of the target protein in cells or tissues.

Multiple labeling : multiple target proteins can be detected simultaneously, and multiple labeling can be achieved by using fluorescent dyes with different wavelengths.

Non-invasive : Observing biological samples in a non-invasive way will not change the structure of the sample.

Application of Immunofluorescence

Protein Localization

The distribution of target proteins in cells, such as cell membrane, nucleus, cytoplasm, etc., can be localized by immunofluorescence.

Cell and Tissue Analysis

Immunofluorescence can be used to analyze the expression of specific proteins in cells and tissue sections.

Co-localization Analysis

The co-localization and interaction of different proteins can be observed by multiple labeling and different colors of fluorescent dyes.

Disease Research

Immunofluorescence is used to detect pathological markers and abnormal proteins in cancer, neurological diseases, immune diseases and other studies.

Alpha Lifetech provides immunofluorescence services to help customers achieve accurate visual analysis of target molecules, help researchers deeply understand cell biological processes and disease mechanisms, and then promote the development of biomedical research and clinical diagnosis.

FAQ

1. In the immunofluorescence experiment, the background signal is strong, which makes it impossible to clearly observe the target molecule.

Solutions : (1) Incomplete blocking : Ensure the use of appropriate blocking solution (such as BSA, normal serum, etc.), and block enough time (30 minutes to 1 hour) to prevent non-specific binding. (2) Antibody concentration is too high : reduce the concentration of primary or secondary antibodies to avoid non-specific binding. Inadequate washing : Increase the number and time of washing to ensure the removal of unbound antibodies. (3) Use high-quality antibodies : select antibodies with strong specificity and high affinity to reduce the possibility of non-specific binding.
2. The fluorescence signal is weak or cannot be detected, resulting in the target molecule is invisible.

Solutions : (1) Antibody concentration is too low : increase the concentration of primary antibody or secondary antibody to ensure that the antibody can fully bind to the target antigen. (2) Poor quality of fluorescently labeled antibodies : Select high-quality fluorescently labeled antibodies to ensure the fluorescence intensity and stability of the dye. (3) Sample fixation problem : Ensure that the fixation steps are sufficient to avoid degradation or conformational changes of the target molecule during the fixation process. (4) Fluorescence attenuation of fluorescent dyes : the use of anti-fluorescence attenuation agent of the sealing solution, to prevent the fluorescence signal attenuation over time.
3. The sample may be contaminated, resulting in inaccurate or unreliable experimental results.

Solutions : (1) Unclean operation : ensure that all the equipment and reagents used in the experiment are clean and avoid cross-contamination. (2) Use fresh reagents : Replace the reagents regularly to avoid unstable results caused by reagent aging or deterioration. (3) Avoid sample drying : Keep the sample moist during the operation to prevent the sample from drying before fluorescence microscopy.
4. The binding of antibody to target antigen is not specific, which may lead to false positive or false negative.

Solutions : (1) Choose high-quality antibodies : Buy a proven, high-quality antibody, or make a fully validated antibody. (2) Optimization of antibody incubation conditions : Adjust the incubation time, concentration and temperature of the antibody to ensure the best binding effect. (3) Negative control : The specificity of the antibody is verified by adding a negative control (e.g., non-immune serum or unrelated antibody).
5. After the fixation step of the cell or tissue, the antibody cannot enter the cell or tissue smoothly.

(1) Appropriate osmotic treatment : Osmotic treatment is performed using surfactants (such as Triton X-100 or Saponin) so that the antibody can enter the cell. (2) Select the appropriate fixative : Ensure that the fixative stabilizes the cell structure without affecting the entry of the antibody.

reference

[1] Im K, Mareninov S, Diaz MFP, Yong WH. An Introduction to Performing Immunofluorescence Staining. Methods Mol Biol. 2019;1897:299-311. doi:10.1007/978-1-4939-8935-5_26

[2] Odell ID, Cook D. Immunofluorescence techniques. J Invest Dermatol. 2013;133(1):e4. doi:10.1038/jid.2012.455

[3] Aros CJ. Indirect Immunofluorescence of Tissue Sections. Methods Mol Biol. 2022;2386:17-26. doi:10.1007/978-1-0716-1771-7_2

[4] Maecker HT, Maino VC, Picker LJ. Immunofluorescence analysis of T-cell responses in health and disease. J Clin Immunol. 2000;20(6):391-399. doi:10.1023/a:1026403724413