Cell Fixation and Permeabilization
Proper fixation and permeabilization are essential for optimal detection. Cells are usually fixed with 2%-4% paraformaldehyde and then permeabilized using agents like 0.1% saponin or 0.02% Triton X-100. Saponin offers a gentler approach but may not be ideal for nuclear antigens, where Triton might be more effective. When using saponin, remember that it creates reversible membrane permeability, so you should re-permeabilize during each antibody incubation step. Alternatively, ice-cold methanol can be used to avoid detergents altogether.
Antibody Specificity
High specificity is crucial in immunofluorescence to avoid non-specific binding and background noise. Use purified antibodies and include appropriate controls, such as secondary-only staining, to validate your results and reduce false positives.
Optimal Antibody Dilution
Adjusting the antibody dilution is key to achieving clear signals without excessive background. A typical range is 1 μg/mL for purified antibodies or 1:100 to 1:1000 for antiserum. If you're new to an antibody or antigen, perform a concentration gradient test to find the best ratio.
Buffer and Blocking Solutions
While PBS works well for many antigens, consider using optimized buffers containing different ions (e.g., calcium, magnesium) for better performance. Rockland offers specialized IHC blocking buffers suitable for fluorescent staining as well.
Choosing the Right Secondary Antibody
For single-color experiments, use pre-adsorbed secondary antibodies. For multi-color staining, always go for pre-adsorbed options from the same species to minimize cross-reactivity.
Appropriate Cell Density
Selecting the right number of cells is important. Too many cells can lead to poor structure and high background, while too few may result in weak adhesion and poor viability.
Multiple Staining Strategies
When detecting two antigens simultaneously, ensure that the primary antibodies come from different sources, have distinct markers, and that the secondary antibodies are consistent.
Reducing Background Noise
To lower background, replace BSA with normal serum from the secondary antibody species. Also, increase washing steps—wash at least three times for five minutes each using PBS + 0.05% Tween.
Cover Slip Application
The final step in immunofluorescence is to cover the sample with a mounting medium that increases refractive index and protects the specimen from photobleaching.
Data Analysis
When analyzing the whole sample, focus on representative cells to ensure accurate and meaningful results.
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