Routine Staining (H&E)
Two dyes, opposite charges
Hematoxylin is the basic dye and stains acidic (basophilic) structures: nuclei, ribosomes, rough endoplasmic reticulum, cartilage matrix. Eosin is the acidic dye and stains basic (acidophilic/eosinophilic) structures: cytoplasmic proteins, collagen (pale), and red cells (bright).
Hematoxylin requires a mordant - aluminum in the alum hematoxylins (Harris, Mayer, Gill). Progressive hematoxylin is stained to its endpoint and needs no differentiation; regressive hematoxylin is deliberately overstained and then differentiated in acid alcohol to remove excess dye. After differentiation a bluing step (Scott's tap water, lithium carbonate, ammonia water) raises pH and turns nuclei blue; skip it and nuclei look gray-brown.
Troubleshooting by appearance
Pale or absent nuclei suggest exhausted hematoxylin, over-differentiation, or an acidic unblued solution. Uniformly blue, hazy slides suggest under-differentiation or precipitate from an oxidized, old hematoxylin - filter it. Weak pink or absent eosin means exhausted eosin or loss during prolonged alcohols, since eosin is washed out by alcohols and by water carried into the dehydrant. Reddish-brown deposits are formalin pigment, not a staining problem. Eosin is available alcoholic or aqueous; the alcoholic form is faster and gives crisper red cells, while over-eosin makes the slide uniformly pink and hides nuclear detail.
Rule of thumb: for reversal questions - hematoxylin (basic dye) stains acidic nuclei blue; eosin (acidic dye) stains basic cytoplasm pink.