There are five important controls for immunofluorescence:
- Positive Control: This is crucial to ensure the staining protocol worked correctly. A positive control is a sample known to express the target protein in abundance. If staining doesn't appear in the positive control, there was a problem with the staining process.
- Omitting the Primary Antibody Control: This control involves incubating the sample with the antibody dilution buffer but without the primary antibody. It reveals whether the signal is due to non-specific binding of the secondary antibody to the sample. This can happen if the secondary antibody aggregates or binds non-specifically.
- Absorption Control: This control demonstrates the primary antibody's specificity. It involves incubating the sample with an immune-depleted primary antibody. The immune-depleted antibody should not produce any signal, confirming that the staining observed with the regular primary antibody is specific to the target protein.
- Isotype Control: This control involves incubating the sample with a non-immune antibody of the same isotype (e.g., IgG1, IgG2a, IgM) as the primary antibody but lacking specificity to the target protein. This helps determine if the staining is due to non-specific interactions of the primary antibody's isotype. The isotype control should show minimal background staining distinct from the specific staining.
- Omitting the Secondary Antibody Control: This control helps identify if the observed fluorescence is from autofluorescence, which is the natural fluorescence of some cells. Tissues like the brain, lungs, and colon are prone to autofluorescence. By omitting the secondary antibody, any remaining fluorescence indicates autofluorescence.
These controls help to validate the staining results and troubleshoot potential issues, ensuring the reliability and accuracy of immunofluorescence experiments.
Echo Bio provides a comprehensive range of isotype controls to support your research.
- Extensive catalog: We offer a wide variety of isotype controls from different species and with various specificities.
- Stringent quality control: Our isotype controls undergo rigorous testing to ensure specificity and performance.
- Technical expertise: Our team can assist you in selecting the most appropriate isotype control for your experimental needs.
Ensure the accuracy and reliability of your antibody-based research with high-quality isotype controls.
3 months ago
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