Special stains
Weigert's iron haematoxylin, Biebrich scarlet–acid fuchsin, phosphomolybdic/phosphotungstic acid and aniline blue give black nuclei, red cytoplasm and muscle, and blue collagen, after an optional Bouin's mordant.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Masson's trichrome
Weigert's iron haematoxylin, Biebrich scarlet–acid fuchsin, phosphomolybdic/phosphotungstic acid and aniline blue give black nuclei, red cytoplasm and muscle, and blue collagen, after an optional Bouin's mordant.
How it differs from the standard method
- Iron haematoxylin resists the acidic dye solutions.
- PMA/PTA time decides the collagen colour.
- Used to stage and quantify fibrosis.
1Asking the tissue one chemical question
H&E shows architecture but leaves much unanswered: is that pink band collagen or smooth muscle, are those clear cells full of glycogen or mucin, is that purple deposit calcium? A special stain is a chemical reaction chosen to colour one class of molecule.
Masson's trichrome
Red dye everywhere, then a polyacid pulls it out of collagen, which takes up aniline blue instead.
PAS
Periodic acid makes aldehydes in glycogen, mucins and basement membranes; Schiff reagent turns them magenta. Diastase removes glycogen.
Alcian blue
A copper phthalocyanine dye that binds negatively charged mucins; the pH decides which ones.
Alizarin red S
Chelates calcium into an orange-red complex; specific only near pH 4.2.
Oil Red O
A dye more soluble in fat than in its solvent; works only on frozen sections.
2Specificity is a matter of rates and conditions
Many special stains work by differences in speed rather than all-or-nothing binding. In the trichrome, phosphomolybdic or phosphotungstic acid displaces the red dye from collagen within minutes but from cytoplasm only over tens of minutes, so the time decides whether collagen is blue, purple or whether everything turns blue.
3Controls and quantification
Every special stain needs a control section known to contain the target, stained in the same run: a failed stain and a true negative look identical. Some stains also need a negative control on the same tissue, most famously the diastase-digested serial section that shows which PAS-positive material is glycogen.
4What survives processing decides the method
Processing through alcohol and xylene dissolves neutral lipid, so Oil Red O needs frozen sections and an aqueous mountant. Decalcifying a block removes the calcium that alizarin is meant to find.
Key terms
Practise these as flashcards →Share of a section's area that stains as collagen, used to quantify fibrosis.
Phosphomolybdic or phosphotungstic acid; displaces small red dyes from collagen so a larger blue dye can bind.
Basic fuchsin decolourised with sulphurous acid; turns magenta when it reacts with aldehydes.
A serial section digested with amylase before PAS; glycogen disappears, other PAS-positive material stays.
Acidic mucins whose charge comes from sialic acid carboxyls or from sulphate esters.
A fat-soluble dye such as Oil Red O that stains by dissolving into lipid rather than binding.
When to use it
- Masson's trichrome or picrosirius red: fibrosis in liver, kidney, lung, heart or skin.
- PAS with diastase: glycogen storage, fungal walls, basement membranes and neutral mucins.
- Alcian blue (often combined with PAS): acidic mucins in gut, airway and cartilage.
- Alizarin red S: calcium in tissue or in osteogenic cell cultures.
- Oil Red O: neutral lipid in frozen sections of fatty liver, adipose tissue or atherosclerotic plaque.
Compared with alternatives
The routine first look; special stains answer the questions H&E cannot.
When the target is a specific protein rather than a class of molecule, for example a collagen type.
Decides what is left to stain: lipids need frozen tissue, calcium an undecalcified block.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Weigert's iron haematoxylin, Biebrich scarlet–acid fuchsin, phosphomolybdic/phosphotungstic acid and aniline blue give black nuclei, red cytoplasm and muscle, and blue collagen, after an optional Bouin's mordant.
- Iron haematoxylin resists the acidic dye solutions.
- PMA/PTA time decides the collagen colour.
- Used to stage and quantify fibrosis.