ESSENTIAL ACCESSORY REAGENTS!

In histological staining, we are obviously concerned with the quality of the hematoxylin and the counterstain, whether we use eosin or the phloxin/safran compound.

However, staining will not be successful if you don't use quality ancillary reagents! When using regressive hematoxylin, it must be differentiated correctly. A weakly acidic aqueous solution of acetic or hydrochloric acid is usually used. The use of an acidified alcoholic solution, which provides a gentler, more gradual and therefore more controllable differentiation, is also frequently employed. Such solutions are available from reagent suppliers in various concentrations. As the differentiation step is critical to obtaining good nuclear staining, and has been carefully checked at the onset, we don't want an error in dilution to jeopardize it! 

There are commercially available ready-to-use or concentrated differentiation solutions that will give you the assurance of reproducibility in your color hues obtained without ruining your budget.

The next step in the staining process is to expose the stained tissue to a slightly alkaline reagent, which will "blue" the sections, turning nucleuses from red to blue. For a long time, naturally alkaline tap water was used for this step, but the changing pH of tap water as the seasons change - for example, as the snow melts in spring - and the advent of several commercial bluing reagents have made this method rather obsolete. Scott's water solutions or buffer formulas meet this need perfectly, while providing reproducibility difficult to achieve by making your own homemade reagents.

In conclusion, if you or your pathologists are not satisfied with your stains, consider taking a look at accessory reagents!

1. What are the main dyes used in classical histological stains?

The main dyes are:
• Hematoxylin for staining nuclei
• Eosin or the phloxine/safran counterstain for cytoplasm and other tissue components.

2. Why is the quality of accessory reagents important in histological staining?

Because good staining depends not only on the primary dyes, but also on the reagents used for differentiation and bluing. Without quality accessory reagents, staining may be poor even if the dyes themselves are excellent.

3. What is the purpose of differentiation in a regressive hematoxylin stain?

Differentiation removes excess hematoxylin to produce sharp and well-defined nuclear staining.

4. What types of solutions are used for differentiation?

• Weakly acidic aqueous solutions (acetic or hydrochloric acid)
• Acidified alcohol solutions, which allow smoother, more controlled differentiation.

5. Why is it preferable to use commercial ready-to-use differentiation solutions?

Because they provide:
• Consistent, reproducible results
• Reduced chance of dilution errors
• Stable control of staining intensity

6. What step follows differentiation?

Bluing, which consists of exposing the section to a slightly alkaline reagent to convert hematoxylin from red to blue.

7. Why is tap water no longer recommended for bluing?

Because its pH varies depending on region and season, leading to inconsistent staining results.

8. What products are now used for bluing?

• Scott’s tap water substitute
• Commercial ready-to-use bluing buffers
These ensure stability and reproducibility.

9. What should be checked if histological stains in a laboratory are unsatisfactory?

It is recommended to evaluate the quality and preparation of accessory reagents (differentiation and bluing solutions), as they greatly influence the final staining outcome.