Washing protocol - Histology

What is the Purpose of Washing in Histology?

In Histology, washing is an essential step in the preparation of tissue samples for microscopic examination. The washing process helps to remove excess stains, fixatives, and other chemicals that can interfere with the visualization of tissue structures. Proper washing ensures that the tissue sections are clean and that the staining is precise and specific.

Why is Washing Important?

Washing is critical for several reasons:

It prevents non-specific staining, which can obscure the tissue details.
It removes residual chemicals that might affect the staining process.
It enhances the clarity and contrast of the stained tissue sections.

When Should Washing be Performed?

Washing should be performed at various stages of the histological process, including after fixation, during and after staining, and sometimes even before mounting the tissue sections on slides. The exact timing and frequency of washes depend on the specific protocols being followed and the types of stains or reagents used.

What Solutions are Used for Washing?

The choice of washing solution depends on the stage of the histological process and the chemicals involved. Common washing solutions include:

Distilled water: Used for general washing to remove water-soluble substances.
Buffer solutions: Used to maintain pH and remove buffer-soluble substances.
Alcohol solutions: Used to remove dehydrants or clearants.
Saline solution: Used in immunohistochemistry to wash away antibodies.

What are the Steps in a Typical Washing Protocol?

While washing protocols can vary, a typical washing protocol might include the following steps:

Initial Rinse: After fixation, rinse the tissue sections with distilled water to remove fixatives.
Intermediate Wash: During staining, wash the sections with buffer solutions between the application of different stains or reagents.
Final Wash: After the staining process is complete, wash the sections thoroughly with distilled water or appropriate solutions to remove excess stains.
Dehydration: Wash the sections with increasing concentrations of alcohol if dehydration is required before mounting.

How Long Should Each Wash Last?

The duration of each wash can vary based on the specific protocol, tissue type, and reagents used. Generally, washes can last from a few seconds to several minutes. It is important to follow the recommended protocol times to ensure optimal results. Over-washing can lead to loss of tissue sections or excessive removal of stains, while under-washing can result in inadequate removal of excess reagents.

What are Common Mistakes to Avoid?

Common mistakes in washing protocols include:

Insufficient washing, leading to non-specific staining or chemical residues.
Over-washing, which can result in loss of tissue sections or essential staining.
Using incorrect washing solutions that may not effectively remove specific reagents.
Not following the recommended washing times, which can compromise the quality of the tissue sections.

Conclusion

Proper washing protocols are vital for the successful preparation of tissue samples in histology. By understanding the purpose, timing, solutions, and steps involved in washing, as well as avoiding common mistakes, histologists can ensure high-quality, clear, and specific tissue staining. Always adhere to the specific protocols recommended for your particular histological procedures to achieve the best results.