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Short answer
Histological staining adds colour to thin tissue sections so cell and tissue structures can be seen under a microscope. Labs sort it into three tiers: routine staining (hematoxylin and eosin, H&E), special stains that target a specific structure or organism, and advanced stains (immunohistochemistry and in situ hybridization).
Definition and scope
Unstained tissue sections are nearly transparent, so a dye is needed to separate nuclei, cytoplasm, fibres and microorganisms. The Leica Biosystems introduction to routine and special staining uses “routine staining” to mean H&E, which is applied to all tissue specimens. “Special stains” are the many alternative techniques used when H&E does not give the information needed. Immunohistochemistry and in situ hybridization are treated as a separate category, “advanced stains”.
This guide is the overview. The stain-specific pages cover the detail: H&E staining, Congo red staining, Ziehl-Neelsen staining, immunostaining and in situ hybridization.
Routine, special and advanced stains
Routine: hematoxylin and eosin
Hematoxylin is a basic dye that colours nuclei and RNA-rich structures such as ribosomes a purplish blue. Eosin is an acidic dye that is typically reddish or pink and colours cytoplasm, cell walls and extracellular fibres. Because the pair is applied to all tissue specimens, it is the baseline every other stain is compared against.
Special stains
Special stains are chosen by what they need to reveal: collagen, fungi, iron, mucins, acid-fast bacteria and so on. According to Leica Biosystems, special stains are harder to automate than H&E because of their variety and complexity.
Advanced stains
Immunohistochemistry and in situ hybridization detect specific proteins or nucleic acid sequences rather than relying on a dye’s chemistry alone. They are a different category from special stains, and the two terms should not be used interchangeably.
Which stain shows what
The table combines the targets listed by Leica Biosystems with the expected results given in Table 7-1 of the “Special studies” chapter of the Manual of Surgical Pathology (PMC). A dash means Table 7-1 has no entry for that stain.
| Stain | What it demonstrates | Reported result |
|---|---|---|
| Hematoxylin and eosin | Nuclei (hematoxylin); cytoplasm and extracellular fibres (eosin) | Purplish blue nuclei; pink to reddish cytoplasm |
| Masson’s trichrome | Collagenous connective tissue fibres; helps separate collagen from smooth muscle | Mature collagen, type 1: dark blue |
| Gomori trichrome | Muscle fibres, collagen and nuclei | Mature collagen, type 1: dark blue |
| Periodic acid-Schiff (PAS) | Carbohydrate-rich structures such as glycogen, glycoproteins, mucus and basement membranes | Glycogen, basement membranes, mucins, colloid and fungi: red |
| Modified Gomori methenamine silver (GMS) | Fungi, basement membrane and some opportunists such as Pneumocystis | Fungi and Pneumocystis: black |
| Perls’ Prussian blue | Ferric (Fe3+) iron, for example hemosiderin | – |
| Ziehl-Neelsen | Acid-fast bacilli, mainly tuberculosis in lung tissue | TB: red and beaded |
| Alcian blue (pH 2.5) | Acidic mucosubstances (mucins); Alcian blue/PAS shows both acidic and carbohydrate-rich material | Acid mucins: blue |
| Congo red | Amyloid | Orange-red, with apple-green birefringence under polarized light |
| Reticulin | Reticulin fibres | Black |
| Verhoeff-van Gieson | Elastic fibres | Blue-black to black |
| Gram stain | Bacteria (gram-positive and gram-negative) | Gram-positive: blue; gram-negative: red |
What makes a stain run reliable
Leica’s introduction gives a short list of factors that decide whether a stain is reproducible. It works as a checklist for lab managers reviewing a staining area:
- Positive control slide. Always use a control slide known to contain the structure or substance the stain is meant to demonstrate.
- Dewaxing first. A solvent removes the paraffin; it is always the first step on a paraffin section.
- Timing. Inaccurate timing gives inconsistent results.
- Washing. Washing steps are frequently the cause of variable results.
- Reagent condition. Some reagents need refrigeration because of fungal or mould growth, light-sensitive reagents are kept dark, and some dye solutions deteriorate slowly while others are made fresh or left to ripen.
- Documentation. Any departure from the method should be recorded.
Tissue sections for any of these stains are cut on a microtome.
Where it matters for labs and supply decisions
According to Leica Biosystems, most clinical labs use fully automated systems for H&E, and manual H&E is now rare. Special stains are less automatable, so larger labs typically automate the common ones and keep a hand-staining area for the rest. For purchasing and storage planning this means two parallel workflows: a high-volume automated H&E line, and a smaller manual area that needs its own reagent inventory, with refrigerated and light-protected storage for the reagents that require it.
Common misunderstandings
Special stain and immunohistochemistry are the same thing
They are not. Special stains rely on dye chemistry for a target; immunohistochemistry and in situ hybridization are the “advanced” category. The method used to demonstrate a protein is described in the immunostaining guide.
H&E is only a basic first look
H&E is the routine stain for all tissue specimens, and special or advanced stains are ordered when it does not give the information needed. It is the starting point of the workflow.
A colour is the same in every method
Colours depend on the method: Congo red amyloid is orange-red, Ziehl-Neelsen TB is red, and Gram-positive organisms are blue. The expected result belongs to the specific stain and should be checked against a control slide, not assumed from another method.
FAQ
What is histological staining?
It is the use of dyes or detection reagents on thin tissue sections so that nuclei, cytoplasm, fibres, deposits or microorganisms can be told apart under a microscope.
What is the difference between routine and special stains?
Routine staining means H&E, applied to all tissue specimens. Special stains are the many alternative techniques used when H&E does not give the information needed.
Are immunohistochemistry and in situ hybridization special stains?
Leica Biosystems places them in a separate category, advanced stains, rather than among the special stains.
Why are special stains harder to automate than H&E?
According to Leica Biosystems, the variety and complexity of special stains make them less automatable, which is why larger labs usually keep a hand-staining area alongside automated H&E.
Why does a stain need a control slide?
A control slide known to contain the target structure or substance shows whether the stain worked, so a missing result on the patient slide can be interpreted.
Sources
- Leica Biosystems, “An introduction to routine and special staining” (knowledge pathway), https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-routine-and-special-staining/, checked 2026-10-29.
- “Special studies”, in Lester SC (ed.), Manual of Surgical Pathology, Elsevier; PMC chapter, https://pmc.ncbi.nlm.nih.gov/articles/PMC7173451, Table 7-1, checked 2026-10-29.
