Cell Fixation and Permeabilization
Proper fixation and permeabilization are crucial for successful immunofluorescence. Cells are typically fixed with 2%-4% paraformaldehyde to preserve their structure. After fixation, permeabilization allows antibodies to enter the cell. This can be done using 0.1% saponin or 0.02% Triton X-100. Saponin provides reversible permeability, so it must be used during every antibody incubation step. Alternatively, ice-cold methanol can be used to avoid detergents altogether.
Antibody Specificity
Highly specific antibodies are essential to minimize background noise and non-specific staining. Purified antibodies often work well, but including appropriate controls—such as secondary-only staining—can help identify true signals and reduce false positives.
Optimal Antibody Dilution
Choosing the right dilution is key. A common starting point is 1 µg/mL for purified antibodies or 1:100–1:1000 for antiserum. If you're new to an antibody or antigen, performing a concentration gradient experiment can help find the best balance between signal strength and low background.
Buffer and Blocking Solutions
While PBS works for many antigens, certain targets may benefit from alternative buffers containing ions like calcium or magnesium. Rockland offers optimized IHC blocking solutions that are also suitable for fluorescent staining experiments.
Choosing the Right Secondary Antibody
For single-color experiments, pre-adsorbed secondary antibodies are recommended. For multi-color staining, always use pre-adsorbed secondary antibodies from the same species to avoid cross-reactivity and ensure accurate detection.
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 uneven staining. Aim for a balanced density for optimal results.
Multi-Staining Techniques
When detecting two antigens simultaneously, ensure the primary antibodies come from different sources and have distinct markers. The secondary antibodies should be from the same species to avoid confusion and ensure accurate visualization.
Reducing Background Noise
A high background is a common issue. To address this, use normal serum instead of BSA for blocking, reduce the antibody concentration, and increase washing steps. At least three washes of five minutes each with PBS + 0.05% Tween are recommended to remove unbound antibodies effectively.
Cover Slip Application
As the final step, applying a cover slip increases the refractive index and protects the sample from photobleaching, ensuring long-term stability and clarity under the microscope.
Data Analysis
When analyzing the entire sample, focus on selecting representative cells for imaging and data collection. This ensures that your results are both accurate and meaningful.
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