Hematoxylin and eosin stain: Head

This is perhaps the most widely used stain for diagnostic histopathology. Hematoxylin is a natural dye that is extracted from the heartwood of the logwood tree Haemotoxylon. Several variations of the hematoxylin solution are available. The final color of hematoxyin is blue to blue-black, depending on the mordant being used. Progressive formula (the stain gets darker with time) and regressive formula (the slide is basically overstained and the intensity is controlled by differentiation) are available. Typically, hematoxylin can only stains tissue at an acidic pH. Hematoxylin at acidic pH, however, does not gives only a weak red-wine like color. When the hematoxylin is subjected to mildly alkaline environment, it will give the blue color. Hematoxylin stains nuclear blue to dark-blue. it also stains the matrix of hyaline cartilage, myxomatous, and mucoid material pale blue. Hematoxylin also stains myelin weakly but the effects is not noticeable if it is combined with eosin stain.
Eosin is the most common counterstain to go with hematoxylin. The intensity of eosin varies with the formula as well as the fixative. Tissues that are fixed in Zenker’s fixative or ethanol are brightly eosinophilic. Eosin stains cytoplasm pink to red; red blood cells are also bright red.
Click thumbnail to see an oligodendroglioma stained with HE stain.

Hematoxylin and eosin stain for frozen sections and cytologic preparation: Head

Since hematoxylin turns blue in alkaline environment, ammonia water or other alkaline solutions such as lithium carbonate are often used for this purpose. In frozen sections and intraoperative cytologic preparations (squash preparations), this alkaline step is strong enough to damage nuclear details on frozen sections and squash preparations of brain and spinal cord tissue.
In addition, necrotic debris tends to fall off at this step. If this step is a problem, it can be eliminated. The slides can be “blued” in running tap water. It will take a little longer but the morphology will also be better.
Click thumbnail to see a cytologic preparation of a metastatic carcinoma in cerebellum stained with HE stain. The large cells are carcinoma cells, the smaller cells are residual internal granular cells.

Toulidine blue stain for intraoperative cytologic preparation: Head

Toluidine blue is a synthetic dye in the thiazins family; stains in this family also include thionin and methylene blue. Toluidine blue is a metachromatic stain that stains nucleus blue and cytoplasm light blue. It is very often used to stain frozen sections and squash preparation of brain parenchymal tissue.
One of the advantages for such application is that toluidine blue stains the mucoid substances of low-grade astrocytoma pale pink-blue (metachromatic reaction) and makes them easily recognizable.

Diff-Quik stain for intraoperative cytologic prepartion: Head

Diff-Quick stain is most helpful in intraoperative cytologic preparations (squash preparations) when a suspicion of lymphoma and leukemia is raised. It provides good morphologic details of lymphoid cells and also allows easy recognition of lymphoglandular bodies which are detached cytoplasmic fragments of lymphoid cells that are seen in most cases of lymphomas.
Click thumbnail to see a cytologic preparation of a Ewing's sarcoma. Please note the cytoplasmic vacuoles (arrow).

Mucicarmine stain: Head

This stain that utilizes Carmine (alum lake) as a stain. It stains epithelial mucin red and is often used to determine mucin production in metastatic carcinoma of the brain. It is also very useful in detecting cryptococcus that has a mucopolysaccharide coat because mucicarmine stains it red.
Click thumbnail to see intracytoplasmic mucin (arrow) in a metastatic adenocarcinoma.

Periodic acid-Schiff (PAS) stain: Head

The principle of this stain is to oxidize the hydroxyl (-OH) group to aldehyde group (-CHO) by the periodic acid. The aldheyde group will react with the Schiff agent to form the red-purple product.
Since hydroxyl groups that could be oxidized to aldehyde groups are present in glycogen, mucopolysaccharides and mucin, PAS reaction is useful in detecting glycogen, mucin, fungus, corporal amylacea, basement membrane, polygucosan bodies and other substances. PAS also stains lysosomes granules red-purple and can be used in recognition of macrophages; this method is not very reliable. Hematoxylin stiain is usually used as counterstain.
This stain is often used to detect fungal organisms and cytoplasmic accumulation of glycogen.
Click thumbnail to see abnormal accumulation of glycogen in McArdle's disease.

Periodic acid-Schiff (PAS) stain with diastase digestion: Head

Diastase and a-amylase digest glycogen into smaller units that would be washed away during processing. By comparing a slide stained by PAS technique with and without diastase digestion could reveal the amount of glycogen. This is a reliable way only if glycogen is well preserved initially. Glycogen can dissolve in water during fixation. The PAS technique serves only as a rough estimate. Quantitative determination of glycogen content and enzymologic studies should be performed if glycogen storage diseases are suspected. Hematoxylin is usually used as counterstain.

Stains for microorganisms: Head

Gomori’s methenamine silver (GMS) and Grocott’s methenamine silver stain: Head

These stains are typically used to detect fungal organisms such as Histoplasma, Candida, Blastomyces and other fungal organism. Cryptococcus without a mucopolysaccharide coat will not be detected by PAS or mucicarmine stain but are detected by GMS or Grocott’s stain. Grocott’s stain is essentially a modification of Gomori’s methamine silver stain.
These stains share a similarity with PAS stain in using chromic acid as an oxidizing agent in the initial step to oxidize the hydroxyl group to aldehyde group. The tissue is then treated with methamine silver solution. Silver ions will be reduced to silver metal by the aldehyde and give a black color. In contrast to PAS stain, chromic acid, however, is a strong oxidizing agent and will oxidize some of the aldehyde group further to subtance that would not be able to react with silver ion. With this token, the background produced by collagen and basement will be suppressed. Light green is used as a counterstain.

Click thumbnail to see broad based budding yeast in a case of blastomycosis.

Ziehl-Neelsen stain (acid-fast stain): Head

This stained involve carbol fuchsin solution as the staining agent and differentiation in acid. It is used typically for the detection of acid-fast bacilli (stained red); methylene blue is used as the counterstain. A modified version of Ziehl-Neelsen stain with prolonged staining at high temperatureis used in the detection of certain lipofuscins in neuronal ceroid-lipofuscinoses (Batten disease), and also to detect nuclear inclusion bodies in chronic lead or bismuth poisoning.

Fite stain, modified for detection of Nocardia: Head

Fite stain is also a carbol fuchsin solution based stain very similar to that of Ziehl-Neelsen stain. Regular Fite stain detects acid-fast bacilli. A modeified Fite stain featured by reduced staining time and decolorization in sulfuric acid is used to demonstrate Nocardia (stained red). It is always important to check with the technical staff if Nocardia is the organism under question.

Warthin-Starry stain for Spirochetes: Head

This is a silver impregnation for spirochetes. In contrast to other silver impregnations, a separate reducing solution, hydroquinone in this case, is employed to reduce the absorbed silver ion to silver metal. Spirochetes are stained black and the background is yellow. This is a tricky stain and it is always important to check the control slide.

Brown-Hopps modified Gram stain: Head

This is used to detect Gram positive and negative bacilli; Gram positive bacilli are stained deep blue, Gram negative bacilli are stained red, background is yellow.

Mucicarmine stain: Head

For demonstration of cryptococcus (see above).

Periodic acid-Schiff (PAS) stain: Head

For demonstration of fungus in general (see above for mechanism).
Click thumbnail to see broad based budding yeast in a case of blastomycosis.
[Click here to see a related case]

Stains for neurons Head

Cresyl echt Violett for Nissl substance (Nissl stain): Head

This stain essentially stains nucleic acids and will stain the Nissl substance and nucleus blue and is very useful in studying cortical architecture and also structure of the deep gray substance and nuclei.

Gallocyanine stain for Nissl substance: Head

Depending on the mordant being used, gallocyanine stain gives a blue or black color. It stains nucleic acids and is used to demonstrate nuclei and Nissl substance.

Stains for myelin: Head

Luxol fast blue- Cresyl violet Stain (Klüver-Barrera stain): Head

Luxol fast blue dye belongs to the sulfonated copper phthalcyanine type and is the alcohol-soluble counterpart of the water-soluble alcian blue. The staining mechanisms probably involve chemical binding of Luxol fast blue with choline-containing compound in paraffin sections. It is probably one of the, if not the, most popular stain these days for the demonstration of normal myelin. Luxol fast blue is also a versatile stain that can combine with many other stains and one of the best combination for use in the study of neuropathology and normal anatomy is Luxol fast blue (for myelin) combined with cresyl violet stain (for neurons). Since Luxol fast blue stain involves a differentiation step and it is a little technically demanding. Luxol fast blue stain requires paraffin sections.
Luxol fast blue stains normal myelin blue; red blood cells are also stained blue. In paraffin sections of peripheral nerve, the myelin sheath has a bubbly appearance and is a result of artifacts.

Click thumbnail to see a cytologic preparation of a luxol fast blue-cresyl violet stain of normal occipital cortex.

Luxol fast blue-Periodic acid-Schiff (PAS)-Hematoxylin stain: Head

This is a good combination because the PAS stain also highlight macrophage. This is a very useful stain in the study of leukodystrophies and demyelinating diseases. Furthermore, the PAS stain can also highlight capillary basement membranes, fungi, and corpora amylacea.
Click thumbnail to see loss of myelination in a case of demyelinating disease. Note the loss of myelin (blue). Macrophages are stained pale pink.

Luxol fast blue-Hematoxylin-Eosin stain: Head

This is a good stain that gives allows simultaneous evaluation of the cytoplasm, nuclei, and the myelin. The eosin, however, may block the subtle details of the Luxol fast blue stain if the procedure is not perfectly done.
Click thumbnail to see loss of myelination in a case of chronic multiple sclerosis as illustrated by luxol fast blue-hematoxylin-eosin stain.

Luxoll fast blue-Oil red O stain for frozen sections: Head

This is a useful stain but is more useful for research than for routine diagnostic work. Since accumulation of lipd is often seen in areas with degenerated myelin such as those seen in long tract degeneration in the spinal cord, this is a very useful stain to demonstrate simultaneously the loss of myelin and lipid accumulation. This stain must be performed on frozen sections.

Luxol fast blue-Holmes stain: Head

Holmes stain is a silver stain that is essentiall a modified Bodian stain that stain axons. The combinatin of Luxol fast blue to demonstrate myelin and Holmes to demonstrate axons is a perfect stain to study demyelinating diseases; they are featured by loss of myelin with preservation of axons.

Weil’s stain: Head

Weil’s stain is a hematoxylin based stain with ferric alum as the mordant. This is also a regressive stain and need differentiation. It stains both myelin and red blood cells dark red. It can be used to demonstrate myelin as well as red blood cells. This method works well with frozen section but also works with both paraffin/celloidin embedded sections.

Marchi’s stain for degenerating myelin: Head

In contrast to Luxol fast blue and Weil’s stain that stains for normal myelin, Marchi’s stain is a stain that uses osmium tetraoxide as the staining agent and stains for degenerated myelin. Potassium dichromate is used as an oxidizing agent to achieve the selective staining property. Marchi’s stain stains degenerating myelin. Abnormal myelin will stain black, normal myelin and other structures will not be stained, unless a counterstain is used. This method needs frozen sections.

Stains for neurofibrils: Head

Bielschowski’s stain: Head

This is a silver impregnation method that involves silver ion complex (formed by adding sodium hydroxide to silver nitrate) and, similar to many other silvers, toning by gold choride. The axons are stained black and the neuronbodies is deep to golden brown. Other cell bodies are also deep to golden brown. This is a very good stain to demonstrate neurofibrillary tangles and senile plaques in Alzheimer’s disease. It is more useful in the CNS. Demonstration of axons in peripheral nerves by silver stain is better done with Bodian stain.

Click thumbnail to see a plaques and tangles as detected by Bielschowski's stain.

Gallyas stain: Head

This is also a silver impregnation method and is of particular value in demonstration of neuronal cytoplasmic inclusions, neuronal nuclear inclusions, and neuropil threads in multiple system atrophy.

Bodian stain: Head

This is also a silver impregnation method that involves silver proteinate compounds and hydroquinone as a reducing agent. It also takes time and need two days to complete. In best hands, this is an excellent method to demonstrate myeinated and non-myelinated fibers in the central and peripheral nervous system; these fibers are stained black. Neuronal bodies and other cellular elements are not stain. It seems to be a very tricky stain and works best with the central nervous system. Immunohistochemistry for neurofilaments is more than often a better choice for the peripheral nerve system.

Click thumbnail to see preservation of axons in a multiple sclerotic plaque.

Holmes stain: Head

This is an essentially a modified Bodian stain and is particularly useful when combined with Luxol fast blue stain (see above).

Stains for glial cells: Head

Gallyas stain: Head

This is also a silver impregnation method and is of particular value in demonstration of glial cytoplasmic inclusions in oligodendroglial cells in multiple system atrophy.

Phosphotungstic acid hematoxylin (PTAH): Head

This stain has been commonly used to demonstrate reactive astrocytes before immunohistochemistry for glial fibrillary acidic protein (GFAP) was available. For this purpose, this stain is more of historic value than of practical value.

Miscellaneous stains: Head

Masson’s trichrome stain: Head

This is a useful stain that stains nuclei deep blue, skeletal and smooth muscles red, collagen and mucin blue. It also stains brain and spinal cord parenchymal tissue dusky pink to red. It is a very useful stain to evaluate fibrosis; it is also a convenient method to study the relation between fibrous tissues in brain parenchymal tissue in malformations such as meningomyelocele. Striations in skeletal muscles also shows up much better in Masson’s trichrome than in hematoxylin and eosin stain. Although it is called a trichrome, four dyes namely hematoxylin, Biebrich scarlet, acid fuchsin, and analine blue are utilized.

Click thumbnail to fibrosis (blue) in skeletal muscle (red).

Verhoeff-Van Gieson stain: Head

Also known as elastic-Van Gieson (EVG) stain. This is a combination of Verhoeff’s elastic stain which is a hematoxylin stain containing ferric chloride and Wright’s iodine solution and Van Gieson stain which contains acid fuchsin, picric acid, and hematoxylin. This stain stains elastic fibers blue-black to black, collagen pale red, other tissue elements yellow, and nuclei blue to black. Fine eastic fibrisl may not be stained by this method. This stain is a useful stain to detect elastic material particularly in vascular malformations.

Reticulin stain: Head

This is a silver impregnation method that stains reticulin fibers black. This is a very useful stain in studying pituitary lesions as well as vascular malformations. This is also a useful stain for infarcted non-neoplastic anterior pituitary tissue and also, viable and infarcted, pituitary adenoma.
Click thumbnail to see reticular fibers in an infarcted pituitary adenoma.
[Click here to see a related case]

Phosphotungstic acid hematoxylin (PTAH): Head

PTAH stain has many modifications. It can be used to stain for reactive astrocytes but this function is replaced by immunohistochemistry for glial fibrillary acidic protein (GFAP). It is also a wonderful stain to demonstrate striations in skeletal muscles and in rhabdomyosarcomas; it stains oncocytic cell as well as fibrinoid necrotic material dark blue.

Congo red: Head

The selective affinity of Congo red to amyloid to give an red-orange staining on bright field microscopy and its apple green birefringence under polarized light have been regarded as a sine qua non for amyloid in light microscopy. To make good observation, however, needs good Congo red staining and also torsion free optics for polarized light. The lineality of the molecule of Congo red and its ability to inteculate in b-pleated sheet configuration of amyloid give this peculiar staining properties.

Thioflavine S: Head

Amyloid stained with thioflavine S gives a greenish fluorescence under fluorescent microscopy. It is more sensitive than Congo red but is also less specific.

Toluidine blue: Head

Toluidine blue has also been used to detect amyloid although it is not widely used. Amyloid stains orthochromatic blue under light microscopy and gives a dark red birefringence under polarized light.

Perls Prussian blue stain (for iron): Head

This stain stains for ferric ion. The section is incubated in a potassium ferrocyanide solution that contains hydrochloric acid. The ferric ion being released by the hydrochloric acid will react with the ferrocyanide to become ferrous ferricyanide which is blue. This is a good way to look for resolved hemorrhage sites in the brain.

Von Kossa stain (for calcium): Head

This is an interesting staining with features similar to mechanism of photography. Sections are exposed to silver nitrate solution and exposed to strong light. The unreacted silver ions are removed by sodium thisosulfate.

Martius Scarlet Blue: Head

This stain is useful for identification of fibrin and fibrinoid necrosis. It stains fibrin red (early fibrin may color yellow and very old fibrin blue); collagen and nuclei are stained blue, muscle is stained red, red blood cells are stained red.

PAS-orange G stain for anterior pituitary cells: Head

This is essentially a PAS preparation that is double stained by orange G to give the orange-yellow color. With this method, the basophil cells are stained magenta, acidophils and red blood cells are stained orange-yellow, the chromophobes are staine pale blue-grey; nuclei are staine blue-black. It role has largely replaced by immunostaining for hormones in the pituitary.

References: Head

Theory and practice of histotechnology. 2^nd edt. By Dezna C. Scheehan and Barbara B. Hrapchak. Battelle Press 1980.

Theory and practice of histological techniques. 3^rd edt. By Bancroft JD and Stevens A. Churchill Livingstone, 1990.

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