Cell fixation and transparency
For optimal detection, the cells need to be fixed and permeable. These steps are critical, and the cells and antigens need to ensure optimal structure and facilitate antibody binding to the antigen. Typically, this is achieved by the following steps: Cells are fixed with 2%-4% paraformaldehyde followed by permeabilization with 0.1% saponin or 0.02% Triton X-100. The former is a gentle treatment, but it may not be effective for nuclear antigens, and Triton is needed. When saponin is used for permeabilization, it should be noted that it causes reversible permeability of the cell membrane, that is, in addition to the initial phase of permeabilization, it is required to be permeabilized in each antibody incubation step. In addition, cells can be fixed and permeable with ice methanol to avoid the use of detergents.
Antibody specificity
Immunofluorescence requires the use of very specific antibodies that avoid high background and undesirable protein localization results. In most cases, the purification of antibodies works well, but the right controls can help pinpoint the antigen. The use of a secondary stain-only stain as a negative control is beneficial in reducing background interference.
Appropriate antibody dilution ratio
The staining is optimized by optimizing the dilution ratio of the antibody, usually 1 ug/ml of purified antibody or 1:100-1:1000 antiserum sufficient to achieve specific staining results. However, under the premise of ensuring low background dyeing, the signal intensity can be increased by increasing the concentration. If the antibody is used for the first time or an antigen is determined, a concentration gradient experiment is strongly recommended.
Optimize buffers and sealers
Although many antigens can be well stained in common Buffers such as PBS, for some antigens of interest, replacing buffers containing different ions, such as calcium, magnesium, potassium, etc., can be greatly improved. . Rockland offers optimized IHC blocking buffers, which are also suitable for fluorescent staining experiments.
Choose the right secondary antibody
If you need to do an immunization experiment, we strongly recommend that you choose a pre-adsorbed secondary antibody for a single staining experiment; if it is a double or even multiple staining, you must use a pre-adsorbed secondary antibody. At the same time, please choose the secondary antibody from the same species.
Use appropriate cell density
Select the appropriate number of cells for staining. When the number of cells is too large, the cell structure is not good, resulting in a deep staining background and low cell density, which may result in poor cell adherence and poor state.
Multiple staining
When two different antigens are detected in the same sample, simultaneous staining can be performed with the respective antibodies, but the source of the two primary antibodies is required to be different, the markers are different, and the source of the secondary antibodies needs to be consistent.
Lower background
High background is a common problem of immunofluorescence. To solve this problem, normal serum from secondary antibody can be used instead of BSA as a blocking solution to reduce antibody concentration, increase the number of washings, and wash at least three times for five minutes. The recommended washing solution is PBS+0.05. %Tween.
Cover
As the final step in immunofluorescence, the refractive index can be increased to protect the sample.
data analysis
When observing the whole sample, select representative cells for data acquisition and analysis.
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