Basic experimental steps:
(1) Cell preparation. For monolayer growth cells, in the passaging culture, inoculate the cells into the pre-positioned petri dish with treated coverslips, remove the coverslips when the cells are close to growing into a monolayer, and wash them twice in PBS; for suspension growth cells, take logarithmic growth cells, wash them twice by centrifugation with PBS (1000 rpm, 5 min), and prepare cell slices by cell centrifugal dumping machine or prepare cell smears directly.
(2) Fixation. Select the appropriate fixative to fix the cells as needed. After fixation, the cells can be stored in azide-containing PBS for 3 months at 4°C. Wash the cells in PBS for 3×5 min.
(3) Permeabilization. Cells fixed with cross-linking agents (e.g. paraformaldehyde) generally need to be permeabilized before incubation with antibody is added to ensure that the antibody can reach the antigen site. The choice of permeabilizing agent should take full account of the nature of the antigenic protein. The time of permeabilization is generally 5-15 min. after permeabilization, the cells are washed with PBS for 3×5 min.
(4) Closure. The cells are sealed with a sealing solution for 30 min.
(5) Primary antibody binding. Incubate at room temperature for 1h or overnight at 4°C. Rinse with PBST 3 times for 5min each time.
(6) Primary antibody binding. Secondary antibody is required for indirect immunofluorescence. Incubate at room temperature away from light for 1 h. Rinse 3 times with PBST, each time rinsing for 5 min before rinsing once with distilled water.
(7) Sealing and detection. One drop of sealing agent was added, the film was sealed and examined by fluorescence microscope.
(I) Cell preparation
Cells used for immunofluorescence experiments can be adherent cells grown directly on coverslips, suspended cells that have been centrifuged and coated or tissue cell suspensions taken from the body that have been centrifuged and coated. Well-adherent cells are usually placed directly onto coverslips during culture, and the use of poorly adherent cells for immunofluorescence experiments should be avoided, as they may be dislodged by subsequent rinsing operations. For the few experiments that require the use of such cells or suspension cells for immunofluorescence observation, it is recommended to use a cell centrifuge to prepare cell slices or prepare cell smears directly.
(II) Fixation and permeabilization
Except for the study of cell surface antigens or unstable antigens can not be fixed, generally should be fixed. The purpose of fixation is threefold:
1) to prevent cells from falling off the slide;
2) to remove lipids that prevent antigen-antibody binding;
3) to make the specimen easy to store.
The principles of specimen fixation are:
①Cannot damage intracellular antigens;
②Cannot agglutinate proteins;
③Should maintain the cellular and subcellular structure;
④After fixation, the permeability should be maintained, in order to ensure that the antibody is free to enter all the cellular and subcellular components to bind to the antigen.
There are a variety of commonly used fixatives, and the appropriate fixative should be selected according to the nature of the antigen under study and the characteristics of the antibody used. Usually fixation methods can be divided into two categories: organic solvents and cross-linking agents. Organic solvents such as methanol and acetone remove lipids and dehydrate the cells, while precipitating cellular structural proteins. Cross-linking agents such as paraformaldehyde usually form intermolecular bridges through free amino acid groups, thus creating a network structure in which antigens are interconnected. Cross-linkers are more likely to preserve the structure of cells than organic solvents, but because cross-linking hinders antibody binding and may reduce the antigenicity of some cellular components, an additional permeabilization step is required to allow antibody access to the specimen. Both fixation methods may denature protein antigens, so antibodies produced using denatured proteins as antigens may be more effective in immunofluorescence.
The most commonly used fixatives are paraformaldehyde and methanol, with ethanol, acetone, and glutaraldehyde also being used for fixation in a few cases. Generally, better results are obtained with acetone, ethanol and high concentrations of formaldehyde for cytoarchitectural antigens, viruses and some enzyme antigens, while cell membrane-associated component antigens are generally fixed with paraformaldehyde. Antigens within organelles and cellular particles are also generally fixed with paraformaldehyde and need to be permeabilized to allow the antibody to reach the antigenic epitope.
The permeabilization step is only needed when detecting intracellular antigenic epitopes, because the antibody needs to get inside the cell to detect the protein. However, if the protein to be detected is a transmembrane protein and its antigenic epitope is in the intracytoplasmic region, then the cell also needs to be permeabilized. Conversely, if the antigenic epitope to be detected is located in the extracellular segment of the membrane protein, no permeabilization is required. Acetone itself is permeabilizing, so permeabilization is not required when using acetone as a fixative. Methanol is also permeabilizing, but there are occasions when methanol is not suitable because some epitopes are very sensitive to methanol. Commonly used permeabilizers are descaling agents such as Triton, NP-40, and Tween 20, Saponin, Digitonin, and Leucoperm, etc. Triton and NP-40 are potent descaling agents that partially dissolve the nuclear membrane of the cell, and are therefore very suitable for nuclear antigen detection. However, it should be noted that if used at high concentrations or for too long, they will damage proteins, which can affect the results. Triton X-100 is the most commonly used permeabilizing agent, but it will disrupt cell membranes, and therefore is not suitable for use with cell membrane-associated antigens. The latter group of depyrogenic agents are much milder and will create pores in the cytoplasmic membrane large enough to allow antibodies to pass through, but will not lyse the cytoplasmic membrane. They are suitable for cytoplasmic antigens, or antigens on the plasma membrane near the cytoplasmic side, as well as for soluble nuclear antigens.
The general procedure is fixation followed by permeabilization, but for the detection of some water-insoluble target antigens it is preferable to permeabilize and then fixate, mainly because many water-soluble proteins can be removed by permeabilization, thus greatly reducing the background and nonspecific signals of immunofluorescence. After fixation, rinse with cold PBS solution and finally with distilled water to prevent spontaneous fluorescence.
(C) Confinement
The purpose of confinement is to reduce the non-specific binding of antibody, the most commonly used confinement agent is 1% BSA, PBS pH 7.5, other optional confinement agents are 1% gelatin, 1% bovine or serum of the same species as the secondary antibody (3-10%), etc. The most commonly used confinement agents are 1% BSA, PBS pH 7.5, and other confinement agents are 1% gelatin, 1% bovine, or the same species as secondary antibody (3-10%).
(D) Antibody incubation
The primary antibody in the direct immunofluorescence method and the secondary antibody in the indirect immunofluorescence method are both fluorescent antibodies, so it is necessary to pay attention to avoiding light when incubating these antibodies. In addition, to ensure binding quality and prevent drying out, antibody incubation should be performed in a wet box as much as possible.
(E) Sealing and fluorescence observation
The fluorescence-labeled cell slices can in principle be observed directly, especially when the slices are not properly sealed. However, in most cases, in order to preserve the results for further observation, imaging, statistical analysis, etc., it is necessary to seal the film. The conventional method is to use glycerin or neutral resin to seal the film, and in order to enhance the effect of sealing, it is often necessary to add special anti-fluorescence quencher when sealing the film.
(F) Specimen preservation
As the stability of fluorescent pigments and protein molecules are relative, with the prolongation of the preservation time, under the influence of a variety of conditions, the labeled proteins may be denatured and dissociated, and lose their proper brightness and specificity. Therefore, it brings certain difficulties to the preservation of the specimen, so the specimen should be observed immediately after fluorescence staining. Due to the availability of well-performing anti-fluorescence quenchers, fluorescently labeled specimens can be stored at low temperatures (4°C or -20°C) for a considerable period of time. In some cases, taking into account the cost of the experiment and the experimental conditions, it is also possible to take an expedient approach, such as fixing the specimen slice and then storing it at low temperature, and then fluorescently labeling it when needed, i.e., staining it as it is used.