Special Stains in Histopathological Techniques

1. Staining of CARBOHYDRATES Periodic Acid Schiff/PAS PAS with Diastase Best Carmine

Langhan’s Iodine method (Carleton’s method)  Oldest stain, considered obsolete  Rapid stain but not a permanent stain as it fades after a few months Fresh Frozen Azure A Metachromatic Stain Alcian Blue Technique

Metachromatic staining Toluidine Blue Staining Combined Alcian Blue – PAS Technique

Mucicarmine Stain Southgate’s Mucicarmine Technique Hale’s Dialyzed (colloidal) Iron Technique Fluorescent Acridine Orange Technique  Disadvantage is that it is temporary and will only last for about 2 hours once the section is mounted

For glycogen For glycogen  For glycogen  Mast cell granules  Fibrin  Mucin  Not specific for glycogen  May also stain amyloid

For glycosaminoglycans Most popular method for general demonstration of acid mucins For glycosaminoglycans 

Demonstration of mucins  Separating acid mucins and neutral mucins For mucus For encapsulated fungi like Cryptococcus neoformans For acid mucins

For acid mucopolysaccharides

Osmic Acid Stain  Not a dye but an unstable oxide  Used as fixative for electron microscopy and in histochemistry Nile Blue Sulfate

Toluidine Blue Acetone Method Borohydride Periodic Schiff (BHPS)

Demonstration of unsaturated fats

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For neutral fats Cholestrin esters and cholestrin fatty acids  Cerebrosides  Fatty acids and soap For sulfatide deposits For gangliosides

3. Staining of PROTEINS Alkaline Fast Green Method Peracetic Acid – Alcian Blue Sakaguchi’s test

For basic proteins especially protamines and histones For cysteine and cysteine For arginine

4. Staining of ENZYMES Gomori Calcium method Gomori Lead method Lead method for 5nucleotidase (Wachsstein and Meisel) Alpha naphthyl acetate method for non-specific esterases Indoxyl acetate method for nonspecific esterases (Holt and Withers) Tetrazolium method for monamine oxidase (Glenner et. al)

For alkaline phosphatase For acid phosphatase For 5-nucleotidase

For non-specific esterases For esterase activity

For monoamine oxidase activity

5. Staining of NUCLEIC ACID 2. Staining of FATS or LIPIDS Sudan Black Sudan IV (Scharlach R) Oil Red O method in Dextrin

For lipids mainly triglycerides For fats

Fuelgen’s techinique for nuclear DNA  Most reliable and specific histochemical staining

For DNA

technique for DNA, best known for chromatin and nucleoproteins Methyl green-pyronin method Acridine Orange fluorescent staining  Most commonly used fluorochrome to demonstrate DNA and RNA

lamps 7. Staining of BONE MARROW and BLOOD ELEMENTS For RNA and DNA For RNA and DNA

Highman’s Congo Red Technique  Method of choice in many laboratories in demonstrating amyloid Krajian’s Amyloid Staining (Modified Bennhold Method) Methyl Violet – Crystal Violet Method Induced Fluorescent Staining with Thioflavine T  Fluorescence may be imparted to amyloid by staining with thioflavine T and exposing the tissue to ultraviolet or quartz iodine

Glycol methacrylate section

For myeloid cells except basophils

8. Staining of MUSCLE and BONE

6. Staining of CONNECTIVE TISSUE Gomori’s Silver Impregnation Van Gieson Masson’s Trichrome Stain Weigert’s Elastic Tissue Stain Orcein (Taenzer-UnnaOrcein) Krjian’s Techinique (employing congo red) Mallory’s Phosphotungstic Acid Hematoxylin (PTAH) method

Rapid Toluidine – Eosin stain for glycol methacrylate section Wright’s – Giemsa – Jenner Stain Peroxidase Reaction for Myeloid cells

For reticulin fibers For collagen For collagen fibers For elastic fibers For elastic fibers Repaid method of staining elastic fibers  Stain for both CNS material and general tissue structures  For muscle striations For amyloid

Modified Gomori’s Trichrome Stain Mallory’s Phosphotungstic Acid Hematoxylin (PTAH) Heidenhain’s Iron Hematoxylin Lissamine Fast Red Tartrazine Schmorl’s Picro Thionin Method

For muscle fibers and collagen For muscle, neuroglia, myelin, collagen Muscle striations, mitochondria, myelin, and chromatin For muscles and bones For bones (lacunae, canaliculi, and bone matrix)

9. Staining of CENTRAL NERVOUS SYSTEM Bielschowsky Technique Bodian’s Stain

For amyloid Sevier Munger Technique For amyloid For amyloid

Cresyl Fast Violet (Nissl) Stain for paraffin sections Weigert Pal Technique of staining Normal

For neurons, axons, and neurofibrils  For nerve fibers and nerve endings  For demonstrating neuritic plaques and neurofibrillary tangles for the diagnosis of Alzheimer’s disease  Foe neural tissues  Commonly used for demonstrating neuritic plaques and neurofibrillary tangles for the diagnosis of Alzheimer’s disease For missle substance, neurons For myelin sheath

Myelin Sheath Kluver and Barrera Luxol Fast Blue Stain for myelin with Nissl Counterstain Luxol Fast Blue – H&E Stain Luxol Fast Blue – PAS – Hematoxylin Stain Weil’s Method Cajal’s Gold Sublimate

For myelin

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Widely used for melanin demonstration

For myelin For myelin For myelin sheath For astrocytes, nerve cells and nerve fibers

Modified holzer’s Method for astrocytic processes 10. Staining of TISSUE PIGMENTS and DEPOSITS

Calcium Dye lake Reaction Von Kossa’s Silver Nitrate Method Lindquist’s Modified Rhodamine Technique

autogenous pigment (brown or black) normally found in the skin and eyes pathological deposition of melanin occurs in benign lesions such as nevus or mole or in melanoma For staining skeletal system in embryos and fetuses For calcium demonstration For staining copper

11. Staining of MICROORGANISMS Perl’s Prussian Blue

Gomori’s Prussian Blue Turnbull’s Blue Reaction for ferrous iron (hemosiderin) Benzidine Nitroprusside Stain Modified Fouchet’s Technique Schmorl’s Ferric Ferricyanide method for reducing substances

Gomori’s Aldehyde Fuchsin Mallory’s Fuchsin Stain Masson Fontane Technique

For hemosiderin – the iron containing pigment of hemoglobin, seen as yellow to brown granules normally found inside the cell. It is the most common hemoglobin derivative Stain for iron pigments For hemosiderin

For hemoglobin and oxidase granules For liver bile pigments 

For argentaffin cells, chromaffin  For thyroid colloid For bile, melanin, lipofuscins – yellow brown to reddish brown pigment produced by slow oxidation of lipids and lipoproteins, it can be found in hepatocytes, cardiac muscle cells, adrenal cortex, and other organs For lipofuscin For hemofuscin pigment For argentaffin granules and melanin – an

Gram Twort Stain Brown and Brenn Ziehl Neelsen Method Wade Fite Technique Auramine – Rhodamine Fluorescent Method Toluidine Blue Stain for Helicobacter Cresyl Violet Acetate Method for Helicobacter Dieterie Method Levaditi’s Wathin-starry Method Modified Steiner and Stainer Technique for Spirochetes Grocott Methamine Silver (GMS) Lendrum’s PhloxineTartrazine Method Rapid Giemsa

For bacteria For bacteria, Nocardia, and Actinomyces For AFB For leprosy bacilli (M. leprae) and Nocardia For Mycobacteria

For Helicobacter For Helicobacter

For Legionella pneumophilia For spirochetes For spirochetes  For spirochetes  Donovan bodies  Fungi and bacteria For fungi For viral inclusions For HBsAg  For blood and bone marrow parasites (Leishmania, Malaria, and Trypanosomes)  Inclusion conjunctivitis  Toxoplasma  Spirochetes and other bacteria