Immunofluorescence technology is based on the principle of antigen-antibody reaction. First, the known antigen is labeled with fluorescein, and then the fluorescent antibody is used as a probe to carry out cell qualitative and localization analysis of the antigen. Because of its strong specificity, high sensitivity and fast speed, it is widely used in scientific research.
Although the technology is not complicated, many freshmen will encounter various problems when starting experiments, so that they cannot get the ideal experimental results. We have accumulated some experience in long-term experiments, and hope to provide some help to everyone.
First of all, the cell density is the first step for a successful experiment. Too high a cell density will cause the cells to be too crowded and the boundaries are not clear, which will not only cause poor cell morphology, but also easily lead to a dark staining background. Similarly, when there are too few cells, it is not only easy to adhere to the wall and difficult to observe, but also difficult to find cells, and because too few cells may not be active enough, non-specific fluorescent staining is prone to occur. Whether using a six-well plate or a confocal dish, the optimal cell density is 75%-85%.
The choice of fixative depends on the subcellular localization of the antigen (membrane proteins, soluble, cytoskeleton-associated proteins, etc.). 3.7%-4% formaldehyde or paraformaldehyde is the most commonly used fixative and is suitable for most proteins.
If you are studying membrane proteins, it is best to use 3.7%-4% formaldehyde. The cytoskeletal components can be studied by methanol fixation. After fixation, a permeabilization step is generally required. Permeabilization is to make holes in the membrane to make it easier for antibodies to enter cells and bind to antigens. The selection of permeabilizers should fully consider the properties of antigenic proteins. Generally, 0.1%-0.2% Triton-X 100 can be used for permeabilization, while methanol fixation generally does not need to be permeabilized. Methanol itself has the effect of permeabilization.
In order to prevent the binding of endogenous non-specific protein antigens, it is necessary to block with blocking solution before the primary antibody incubation, which can reduce non-specific background staining. The blocking solution can be selected from serum with the same source of secondary antibodies. Generally speaking, serum is more expensive and can be replaced with 1%-5% BSA. BSA can be said to be a universal blocking serum.
In addition, the blocking time should not be too long, 30-60 minutes, and no washing is required after blocking, and the primary antibody can be directly incubated.
After blocking, primary antibody incubation is required. You can choose 4 degrees overnight incubation or room temperature for 3 hours. In our experience, primary antibody incubation at 4 degrees overnight is better, and the antigen-antibody binding will be more sufficient.
The dilution ratio of the primary antibody and the secondary antibody can be optimized according to the specific experimental requirements of the antibody. If the antibody concentration is too low, the signal will be too weak, and if the antibody concentration is too high, the background staining may be too strong. If double immunolabeling is used, the primary antibody should be from different species, and the spectrum of the fluorescent secondary antibody should be separated.
In the immunofluorescence process, there are many washing steps, and PBS is sufficient. During the washing process, firstly, pay attention to gentle movements, the fixed cells are relatively fragile, if too much force, it is easy to blow off the cells, secondly, the washing time should be well controlled, about 5 minutes each time, and thirdly, remember not to dry the cells after removing the solution, otherwise it will easily cause background staining.
