What is immunofluorescence?

Immunofluorescence (IF) technology is based on the principle of antigen-antibody reaction, first labeling known antigens or antibodies with fluorescent groups, and then using this fluorescent antibody or antigen as a probe to detect the corresponding antigens or antibodies in cells or tissues. It is a technology established on the basis of immunology, biochemistry and microscope technology.

The principle of immunofluorescence

The basic reaction of immunology is the antigen-antibody reaction. Because the antigen-antibody reaction has a high degree of specificity, when an antigen-antibody reaction occurs, as long as one of the factors is known, the other factor can be detected. Immunofluorescence technology is to label antibodies (or antigens) with fluorescent pigments that do not affect the activity of antigens and antibodies, and combine with the corresponding antigens (or antibodies) to present a specific fluorescence reaction under a fluorescence microscope.

The main classification of immunofluorescence

Direct method:
The labeled specific fluorescent antibody is directly added to the antigen specimen, stained at a certain temperature and time, and the excess fluorescent antibody that has not participated in the reaction is washed away. After drying at room temperature, the slide is mounted and microscopically inspected.

Indirect method:
After the specific antibody reacts with the corresponding antigen in the sample, the second antibody (anti-antibody) labeled with fluorescein is combined with the first antibody in the antigen-antibody complex to form the antibody-antigen-antibody complex. after washing, the specific fluorescence was observed under fluorescence microscope, then the unreacted labeled antibody was washed, dried and sealed for microscopic examination.

Steps of immunofluorescence

1. Section preparation: fresh tissue is recommended, otherwise the internal structure of tissue cells is destroyed and the antigen is easily diffused. Choose clean and sharp blades, tissue must be frozen moderately (frozen sections), etc., to prevent fragmentation and shedding;
2. Tissue section fixation: immediately after slicing and air-drying, fix 5-10 min with fixed liquid, especially the white film that should be preserved for a long time, must be fixed in time and preserved properly;
3. Permeability: Cells fixed with a cross-linking agent (such as paraformaldehyde) generally need to be permeabilized before incubation with antibodies to ensure that the antibodies can reach the antigen site. The choice of permeabilizing agent should fully consider the nature of the antigen protein. The permeabilization time is generally 5-15 min. After permeabilization, wash with PBS for 3×5 min;
4. Serum blocking: In order to prevent the binding of endogenous non-specific protein antigens, it is necessary to block with serum (same source as the secondary antibody) before incubating with the primary antibody to reduce the background coloration. The time of serum blocking can be adjusted, generally 10-30min;
5. Primary antibody incubation: The most important in immunohistochemical reaction, including incubation time and antibody concentration. There are several primary antibody incubation temperatures: 4 degrees, room temperature, 37 degrees, of which 4 degrees are better; incubation time: this is related to temperature and antibody concentration, generally 37 degrees 1-2 h, 4 degrees overnight (take out from the refrigerator After reheating at 37 degrees for 45 minutes). Specific conditions need to be explored by yourself;
6. Secondary antibody incubation: generally 30 min-1 h at room temperature or 37 degrees, the specific time needs to be explored by yourself, and the reaction must be protected from light. But in immunofluorescence, we generally set the concentration of the secondary antibody and the incubation time first, and then explore the concentration of the primary antibody and the incubation time. Finally, as the storage time of the fluorescein-labeled secondary antibody is prolonged, there may be a large amount of free fluorescein residue. It is necessary to pay attention to small packaging and appropriate centrifugation during preparation;
7. Restaining: the purpose is to form a cell profile so that the target protein can be better located. DAPI double dyeing is commonly used;
8. Sealing: for long-term preservation, generally use buffer glycerin and other sealing film, in addition to a special anti-fluorescence quenching sealing solution. Be careful to avoid bubbles;
9. Section cleaning and fluorescence microscope observation: In order to prevent non-specific staining caused by reagent residues such as primary and secondary antibodies, it is particularly important to properly strengthen the cleaning (extend the time and increase the number of times). Generally, the cleaning before the primary antibody incubation is 3 min* 3 times, and the washing after the primary antibody incubation is 5 min * 5 times.

Common technical problems and solutions in immunofluorescence steps

Darker background staining
a: No blocking of non-specific binding or insufficient blocking
Use non-immune serum from the animal sourced by the secondary antibody for blocking; improve blocking conditions;
b: The concentration of the primary and secondary antibodies is too high
It is recommended to establish a concentration gradient pre-experiment to find the best antibody concentration in a single staining system;
c: insufficient washing
Add 0.2% Tween 20 to the washing buffer for sufficient washing;
d: Cell culture is too dense
Optimize cell growth conditions and find the best seeding density.

Weak or no signal
a: There is no target protein in the target cell
It is recommended to make a positive control, prepare a cell lysate, and use Western Blotting to verify whether the target protein is expressed in the cell;
b: The protein is located in the cytoplasm or nucleus
Antibody cannot penetrate the plasma/nuclear membrane, add a permeabilizing agent to the blocking solution and antibody diluent;
c: Not enough primary antibody binds to the target protein
Extend the incubation time and increase the antibody concentration;
d: The fixation step masks the epitope recognized by the antibody (using formalin and paraformaldehyde fixative)
Shorten the fixation time and replace the fixative.

Fast fluorescence quenching
a: The characteristics of fluorescein itself, poor light stability
Use fluorescein secondary antibody with good light stability;
b: Mounting tablet
Use anti-fluorescence quencher to mount the slide.

Autofluorescence
a: fixed with glutaraldehyde.
Fluorescence quenching was performed before fluorescence staining after fixation, such as NaIO4,NaBH4, glycine, etc., and autofluorescence was examined before staining.
b: it could be autofluorescence.
A negative control was set up to eliminate the background staining by reducing the parameters of the fluorescence microscope.
c: there are high levels of autofluorescence molecules (such as riboflavin, cytochrome, etc.) in cellular components.
High ratio of dead cells to living cells in cultured cells.

Summary: Because there are many operating steps, and at the same time, it is impossible to distinguish non-specific recognition based on the molecular weight like WB when analyzing the results. Therefore, to obtain a perfect immunofluorescence experiment result, in addition to the need for high-quality antibodies, and the experimental conditions In addition to repeated optimization, rigorous experimental controls must be set up. In short, every step of the immunofluorescence experiment from cell sample processing, fixation, blocking, antibody incubation to final mounting and observation and photographing is very critical. It is necessary to strictly control the quality of each step in the experimental process in order to finally achieve your experimental purpose.

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