Routine (H&E) Staining
Dye chemistry
Hematoxylin is a basic (cationic) dye that colors nucleic acids blue-purple; it must be oxidized to hematein and requires a mordant, usually an aluminum alum. Harris hematoxylin is chemically ripened and must be filtered; Gill's uses propylene glycol and is more stable; Mayer's is also alum based. Eosin is an acidic (anionic) dye that stains cytoplasm, collagen and muscle pink to red.
The staining sequence
In regressive staining the slide is overstained, then differentiated in acid alcohol (about 1% HCl in 70% ethanol) to strip dye from cytoplasm, then blued in a weakly alkaline solution such as Scott's tap water, lithium carbonate or ammonia water. Bluing is where most failures occur: acidic tap water leaves nuclei pale or muddy. Progressive hematoxylin needs no differentiation step.
Traps that appear in questions
- Reversing the charges: hematoxylin is basic and stains nuclei (basophilic); eosin is acidic and stains cytoplasm (eosinophilic).
- Distinguishing bluing failure from over-differentiation: both give pale nuclei, but only bluing failure responds to alkaline pH.
- Carryover and contamination: eosin floating into hematoxylin, or water baths with debris.
- Exhausted hematoxylin: confirmed by a control slide, not by adding more stain.
Troubleshooting logic: pale blue nuclei mean check ripening and bluing pH; red-brown nuclei mean the solution is too acidic; weak eosin means over-differentiation or dye exhaustion; precipitate means unfiltered hematoxylin or hard water.
Rule of thumb: Control the bluing step first. If the control slide shows crisp blue nuclei and the test slide is pale, suspect that slide's differentiation time rather than the whole stain set.