What is a Negative Control?
A
negative control refers to a sample in an experiment that does not receive the treatment or test being applied to the experimental group. In the context of
histology, it is a tissue sample processed alongside the test samples but without the primary antibody or specific reagent. This control helps in identifying any
non-specific binding or background staining, ensuring the reliability and specificity of the experimental results.
Why is Negative Control Important?
The implementation of a negative control is crucial for several reasons:
1.
Validation of Specificity: It confirms that the staining or signal observed in the test samples is due to the specific binding of the primary antibody to the target antigen.
2.
Background Noise Identification: It helps in identifying and quantifying any background staining or non-specific binding.
3.
Quality Control: It serves as a quality control measure to assess the overall quality and reliability of the histological procedure.
How is a Negative Control Prepared?
The preparation of a negative control involves processing a tissue section in parallel with the test samples but omitting the primary antibody. The steps typically include:
1.
Fixation and Embedding: The tissue is fixed and embedded in a medium like paraffin, similar to the test samples.
2.
Sectioning: Sections are cut and placed on slides.
3.
Blocking: Non-specific binding sites are blocked using a blocking solution.
4.
Omission of Primary Antibody: Instead of applying the primary antibody, a diluent or buffer is used.
5.
Secondary Antibody and Detection: The sections are then treated with the secondary antibody and detection reagents as per the protocol.
Common Issues and Troubleshooting
Several issues can arise when using negative controls:
1. Unexpected Staining: If staining is observed in the negative control, it indicates non-specific binding or issues with the secondary antibody.
* Solution: Optimize the blocking step or ensure the secondary antibody is specific.
2. High Background: Excessive background staining can obscure results.
* Solution: Check the washing steps, optimize antibody concentrations, and ensure proper blocking.
3. No Signal in Test Samples: This could be due to a problem with the primary antibody or the antigen.
* Solution: Validate the primary antibody and check the tissue processing steps.Applications in Diagnostic Histology
In diagnostic histology, negative controls are indispensable. They are used extensively in:
1. Immunohistochemistry (IHC): To confirm the specificity of antibody-antigen interactions.
2. In Situ Hybridization (ISH): To ensure that the probe hybridization is specific to the target nucleic acid.
3. Fluorescence Microscopy: To verify that the observed fluorescence is due to specific staining and not autofluorescence or non-specific binding.Conclusion
Negative controls play a fundamental role in histological studies by ensuring the specificity and reliability of staining procedures. They help in identifying non-specific binding and background noise, thereby validating the experimental results. Proper preparation and interpretation of negative controls are essential for maintaining the quality and accuracy of histological analyses.
