Why are Controls Important in Histology?
In
Histology, controls are essential to ensure the reliability and accuracy of experimental results. They help distinguish between specific and non-specific staining, validate the staining process, and provide a benchmark for comparison. Without proper controls, interpretations can be misleading, leading to incorrect conclusions and affecting downstream applications.
Types of Controls in Histology
There are several types of controls commonly used in histological procedures:Positive Controls
Positive controls are samples known to express the antigen or structure of interest. They confirm that the staining protocol is working correctly. For instance, if you're staining for a specific protein, a tissue sample known to express that protein would serve as a positive control.
Negative Controls
Negative controls are samples known to lack the antigen or structure of interest. They help identify any non-specific background staining. In immunohistochemistry, a negative control might be a sample treated with an isotype-matched control antibody instead of the primary antibody.
Internal Controls
Internal controls involve using parts of the same tissue specimen that are known to be positive or negative. These controls help validate the staining within the same section, ensuring consistency across the sample.
Key Questions to Consider
What is the Purpose of the Experiment?
Understanding the objective of the experiment is crucial. Are you identifying the presence of a particular antigen? Are you comparing expression levels across samples? The purpose will guide the choice of appropriate controls.
What Tissues or Cells are Being Used?
The type of
tissue or cells used can influence control selection. Different tissues may have varying levels of background staining, necessitating the use of tissue-specific controls.
Are the Controls Processed Identically?
All controls should be processed in the same way as the experimental samples. This includes fixation, sectioning, staining, and other preparatory steps. Identical processing ensures that any differences observed are due to the biological variable and not the experimental procedure.
Are Reagents Consistent?
Consistency in
reagents is vital. Using the same batches of antibodies, stains, and other chemicals can minimize variability and enhance reproducibility. Documenting reagent details and lot numbers can be helpful for future reference.
How Will Results be Interpreted?
Interpreting histological results involves comparing experimental samples to controls. Clearly defined criteria for positive and negative staining should be established beforehand. Photographic documentation of control staining can provide a visual benchmark for comparison.
Implementing Controls in Different Techniques
Immunohistochemistry (IHC)
In IHC, using both positive and negative controls is critical. A positive control ensures that the primary antibody can detect the antigen, while a negative control helps identify any non-specific binding. Including an isotype control can further verify the specificity of the antibody.
Histochemical Staining
For
histochemical staining, such as Periodic Acid-Schiff (PAS) or Hematoxylin and Eosin (H&E) staining, controls should include tissues known to react with the staining procedure. This helps confirm the effectiveness of the staining protocol and the quality of the reagents used.
In Situ Hybridization (ISH)
In ISH, using a sense probe as a negative control can help distinguish between specific hybridization signals and background noise. Positive controls should include tissues known to express the target RNA or DNA sequence.
Conclusion
Employing proper controls in histology is fundamental to obtaining accurate and reliable results. By carefully selecting and processing controls, researchers can confidently interpret their findings and advance our understanding of tissue structure and function. Consistency, thorough documentation, and a clear understanding of experimental objectives are key to successful histological analysis.
