Sometimes the problem with an IHC counterstain isn't the staining time. The hematoxylin itself is simply too strong.
This becomes especially obvious in very cell-dense tissues, such as tonsil or lymph node, and in samples with heavy immune-cell infiltration. A strong nuclear counterstain can make DAB look much dimmer than it really is.
That is particularly problematic when the target is low-expressing or patchy, or when the readout is a discrete point signal, as in ISH or isPLA. In those cases, reducing the hematoxylin intensity can improve signal visibility much more effectively than simply shortening the staining time.
You can dilute it with water, but that also changes the chemical environment of the stain and diluted hematoxylin may become less stable or develop precipitate.
For Mayer's hematoxylin, I prefer diluting it in an acidic vehicle closer to its native formulation.
Mayer-type citric acid diluent
1 L
- Citric acid - 1.0 g
- Distilled water - to 1,000 mL
Mix until completely dissolved.
This corresponds to approximately 5.2 mM citric acid.
Classical Mayer's hematoxylin formulations contain approximately 1 g/L citric acid, together with hematoxylin, an aluminium mordant, oxidant and other components.
This simple diluent therefore preserves the citric-acid concentration while reducing the concentration of the stain.
Important: This is a practical Mayer-type diluent, not a complete recreation of the chemical environment of finished Mayer's hematoxylin. Alum and the other components of the original stain also influence its pH and chemistry.
Diluting Mayer's hematoxylin
| Hematoxylin concentration | Mayer's hematoxylin | Citric acid diluent | Final volume |
|---|---|---|---|
| 50% | 50 mL | 50 mL | 100 mL |
| 20% | 20 mL | 80 mL | 100 mL |
| 10% | 10 mL | 90 mL | 100 mL |
For IHC counterstaining, 10% or 20% hematoxylin for 1-2 min is a useful place to start when undiluted hematoxylin overwhelms a weak DAB signal.
Staining time still needs to be optimized for the tissue, hematoxylin formulation and desired contrast.
What should the diluted hematoxylin look like?
The appearance depends on what you dilute it with.
Diluted with water: violet
If you dilute Mayer's hematoxylin with distilled water, the solution may become violet.
That does not mean it has suddenly stopped working. It can still be used for counterstaining, but water changes the acidity and ionic environment of the original stain and diluted hematoxylin may become less stable or develop precipitate.
I would therefore treat water-diluted hematoxylin as a fresh, preferably one-time-use solution rather than preparing a bottle for repeated use.
Diluted with citric acid: bright red
When Mayer's hematoxylin is diluted in the citric-acid vehicle above, the solution should remain bright red.
That is OK. That is how it should look.
In practice, a citric-acid-diluted staining bath can be used repeatedly for approximately 1–2 weeks, provided it remains clean and performs consistently.
But don't count only calendar days. Count the dilution and workload as well. A 10% bath used for many baskets of slides is not equivalent to a 50% bath used twice.
Keep track of how many baskets or staining runs have been dipped, and replace the solution when staining becomes weaker, inconsistent, contaminated or develops excessive precipitate.
Always standardize the bluing step
If you are comparing hematoxylin concentrations or staining times, keep the next step constant.
Always counterstain using a standardized bluing agent.
Tap water varies in pH and composition, so changing both the hematoxylin and the bluing conditions makes optimization unnecessarily difficult.
Use the same defined bluing solution, concentration and timing each time - for example sodium bicarbonate, lithium carbonate or Scott's tap water substitute.
The goal in IHC is not to make the nuclei as dark as possible.
It is to make them dark enough to provide context without hiding your signal.
References
- Mayer P. Classical Mayer's haemalum formulation: hematoxylin, alum, sodium iodate, citric acid and chloral hydrate.
- Cold Spring Harbor Protocols. Mayer's Hematoxylin. Mayer-type formulation containing approximately 1 g/L citric acid.
- Kiernan JA. Histological and Histochemical Methods: Theory and Practice. Hematoxylin chemistry, mordants and progressive alum hematoxylin formulations.
- Gill GW. Literature on alum hematoxylin formulation, staining strength and progressive nuclear staining.