stains in pathology

Staining of lipids ----------

m.c. method of demonstration of tissue lipids ---------- fresh frozen section

Non polar lipids

Unconjugated lipids §  Fatty acids------ oil red O, sudan black, Sudan III, IV , cooper rubeanic acid §  Free Cholesterol--------filipin , Per-chloric acid naphthoquinone(PAN) Esters §  Cholesteryl esters------Schultz §  Monoglycerides & triglycerides------- Calcium Lipsae

Polar lipids

Phospholipids §  Glycerophospholipids/phosphoglycerides/Glycerol bases o   Phosphatydylcholine/Lecithin --------- Nile blue sulfate, Gold hydroxamate o   Phosphatydylserine--------- Nile blue sulfate o   Phosphatidylethonolamine/Cephalins------------- Plasmal reaction, Gold hydroxamate §  Sphingosine bases o   Sphingomyelins------ferric hematoxylin §  Glycolipids o   Cerebrosides---------PAS o   Sulfatides---------Acetone/ Toluidine blue o   Gangliosides------PAS,Borohydride periodic Schiff(BHPS)

all lipids can be stained by ------- bromine sudan black

unsaturated lipids------- UV-Schiff

Lipofuschins ---- sudan black, Autofluoroscence

Stain for phospholipids-------Nile blue sulfate

Phosphoglycerides(lecithins & cephalins)-------------- gold hydroxamate

Lipids

·         Routine stains

o   Oil red O

o   Sudan black B --------- most sensitive lipid stain

o   Bromine acetone

o   Sudan IV------- m.c. used

o   Sudan black

o   Nile blue sulfate

·         Free fatty acids------------Cooper rubeanic acid method

·         Free Cholesterol -------------Per-chloric acid naphthoquinone(PAN)

·         Proteoglycerides

o   Gold hexamine method

o   Fillipin method

o   UV-Schiff method

o   Osmium tetroxide method

·         Triglycerides --------------Calcium lipase method

OIL SOLUBLE stains /LYSOCHROMES

§  Sudan black is the most sensitive lipid soluble dyes

§  Sudan black--------- greater affinity for phospholipids than other lysochromes-------- coloring neutral lipids/ triglycerides

§  Sudan black B stains --------- phospholipids, neutral lipids

§  Sudan IV / Scharlach R ------- triglycerides/neutral lipids, lipoproteins

§  Oil red O

§  Sudan III -------------- triglycerides , CNS tissues ,

PAS reactive cells and tissue components

§  Glycogen

§  Starch

§  Mucin

§  Basement membranes

§  Alpha-antitrypsin

§  Reticulin

§  Fungi capsule

§  Pancreatic zymogen granules

§  Thyroid colloid

§  Corpora amylacea

§  Russell bodies

Staining of carbohydrates

m.c.  used techniques for carbohydrates demonstration  ---------- PAS

Glycogen

§  PAS

§  Best’s carmine

§  Hexamine silver method

§  Langhan’s iodine

§  Diastase sensitive

mucopolysaccharides---------- Mucin stains

§  PAS

§  Alcian blue

§  Alcian blue-PAS technique

§  Dialysed iron-prussian blue technique

§  Hale’s technique

§  Mucicarmine -------- very specific for epithelial mucins

§  Azure A

§  Colloid iron

§  Toludine blue

§  Southgate’s mucicarmine

The types of mucopolysaccharides are as follows:

Neutral mucopolysaccharides	in glands of the GI tract, in prostate	PAS
Acid (simple, or non-sulfated)	mucins of epithelial cells containing sialic acid	PAS Alcian blue at pH 2.5 Colloidal iron Metachromatic dyes.
Acid (simple, mesenchymal)	contain hyaluronic acid and are found in tissue stroma They can be found in sarcomas.	Alcian blue at pH 2.5 Colloidal iron Metachromatic dyes Digestion with hyaluronic acid
Acid (complex, or sulfated, epithelial)	found in adenocarcinomas.	PAS Alcian blue at pH 1 Colloidal iron Mucicarmine Metachromatic stains
Acid (complex, connective tissue)	in tissue stroma, cartilage, and bone and include substances such as chondroitin sulfate or keratan sulfate.	Alcian blue at pH 0.5.

Most specific mucin stain----------mucicarmine, but it is very insensitive, so it is not really very useful.

Most sensitive stain for mucin --------- PAS

Colloidal iron stains -----------unpredictable.

Alcian blue stains------------ simple, but have a lot of background staining.

	Stains
Acridine orange	Nucleic acid selective fluorescent cationic dye useful for cell cycle determination
Aldehyde Fuchsin	pancreatic islet beta cell granules elastic fibers-- purple/black.
Alician Blue Copper in stain produces blue color.	Acid mucins(sulfomuciins,sialomucins) /mucosubstances---- blue (Nuclei will stain---- pink/red. Cytoplasm ------ lighter pink. ) Proteoglycans Hyaluroonic acid
Alizarin Red S anthraquinone derivative	calcium in tissue
Alkaline Phosphatase	endothelial cells (Sites of alkaline phophatase activity will appear red. Nuclei will stain blue)
Azure A	Acid mucins Proteoglycans
Bielschowsky Stain Silver is used in this histology staining process	reticular fibers neurofibrillary tangles-----black senile plaques------- stain black.
Colloid iron	Proteoglycans Hyaluronic acid Acidic mucins Phospholipids Free nucleic acids Collagen Muscle
Cajal Stain	nervous tissue.
Congo Red	Amyloid-------- orange / red
Conklin staining Uses malachite green	True endospores
Cresyl Violet a Nissl stain	both neurons and glia (acidic parts of cells such as ribosomes, nuclei and nucleoli)
Giemsa Stain Romanowski type stain. Methylene blue and eosin are used.	peripheral blood smears bone marrow parasites and malaria Erythrocytes stain pink/red Platelets and leukocytes stain blue acidic components of the cell(cytoplasm & chromatin) pick up -----basic methylene blue
Golgi Stain	Neurons
Fontana-Masson stain uses silver	argentaffin granules and melanin black (The nucleus will stain pink/red. The cytoplasm will stain light pink.)
Gomori Trichrome mixture of three dyes	connective tissue and collagen green or blue Nuclei will stain gray/blue/black. muscle, keratin and cytoplasm red
H&E	Hematoxylin—basic dye Hematoxylin binds acidic nucleic acids---- blue-purple Eosin—acidic dye----- binds basic/-vely charged structures---- cytoplasm/muscle/connective tissue cationic amino groups on proteins---- pink/orange/red With an H&E stain, mucus and cartilage will stain a light blue color.
Luna Stain	elastin and mast cells
Iron Hematoxylin	stain nuclei bluish/black.
Luxol Fast Blue	Myelin—blue black Neurons ------- violet. Red blood cells-------- blue.
Mallory Trichome	connective tissue to visualize collagen and reticular fibers
Movat's Pentachrome Stain	connective tissue
Masson Trichome. uses light green, iron hematoxylin, and acid fuchsin.	differentiating cellular from extracellular items on connective tissue. Collagen fibers ----- green or blue Muscle and keratin ---- red. Cytoplasm ----- pink to red. Nuclei ------ black
Mucicarmine	Acidic epithelial mucin---deep red
Nissl Stains------ basic dyes	RNA and DNA Nissl body in neurons--------purple/blue.
Nuclear Fast Red	stains nuclei red cytoplasm will be unstained or yellow
Oil Red O This is a histology stain used for	Lipids---------- stain red. Nuclei ---------- stain blue/black.
Orcien Stain	elastin fibers
Osmium Tetroxide Used in optical microscopy to stain lipids	Lipids--black Collagen--- brown RBC—brown Myelin--- black
Periodic Acid-Schiff (PAS)	Glycogen Glycoproteins Proteoglycans Glomeruli Basement memnbraes Mucin Mucoproteins
Phosphotungstic Acid-Hematoxylin (PTAH)	Striated muscle fibres---blue Mitochondria-blue RBC-----blue Nucleus------- blue
PicroSirius Red (polarized)	Collagen
Prussian Blue	Ferric iron Ferritin
Reticular Fiber Stain uses silver salt.	Reticular fibres---- black
Romanowsky Stains ·         Wright's stain, ·         Giemsa stain ·         Jenner's stain ·         Leishman stain These histology stains are based on a combination of eosin and methylene blue.	Blood Bone marrow
Safranin O Safranin O is sometimes used as a counterstain.	Mucin Cartilage Mast cells
Silver Stains	Argyrphilic tissue-black Melanin--- black Reticular tissue—black
Sudan Stains ·         sudan black, ·         sudan IV, ·         oil red O.	Lipids Phospholipids
Tartrazine	Cytoplasm-----yellow Cartilage---yellow RBC-yellow
Toludine Blue	Knee joint growth plate -------- purple
Van Gieson sometimes used in conjunction with iron hematoxylin. used to differentiate collagen and smooth muscle.	Cytoplasm-------- brown/yellow Muscle=== brown/yellow Collagen---red RBC------- yellow Cartilage----pink
Verhoeff Stain	Connective tissue Elastic fibres---- dark brown/black Lung tissues & arteries
Von Kossa Stain	Calcium/calcium deposits Chondrocytes------ black
Wright's	Blood smears Bone marrow RBC—red/pink Eosinophilic granules--- bright orange/red Basophilic granules----- blue/black Cytoplasm of lymphocytes------- pale blue Platelets------ purple

Routine hematoxylin & eosin stain

§  Hematoxylin (blue & basic)--- stains negatively charged structures----- DNA & RNA

§  Eosin (pink & acidophilic)---------stains +ively charged structures --------- mitochondria

Protein staining

·         Alkaline fast green --------- basic proteins (Histidine, Protamines)

·         Paracetic acid- alcian blue------- cystine, cysteine, Arginine

·         Ninhydrin Schiff

·         Millon’s reaction

·         Diazotization coupling method for tyrosine

·         Performic acid alcian blue method for sulfide/disulfide bonds

·         DMB nitrite method

·         Modified sakaguchi method for arginine

DNA staining & demonstration

·         Fluorescent staining

·         FEULGEN reaction

·         In situ hybridization ----------- most sensitive

·         PCR

·         Naphthoic acid hydrazine fuelgen method

RNA staining & demonstration methods

·         PCR

·         Fluorescent staining

·         Methyl green pyronine method

DNA & RNA

·         Gallocyanin chrome alum

·         Acridine orange

Enzyme staining methods

§  Gomori calcium-------- ALP

§  Gomori Pb--------- ACP

§  Lead method---------- 5’N

§  Indoryl acetate------non-specific esterase

§  ACHase-----------?

Connective tissue stains

Commonly used stains

·         Masson’s trichrome

·         Von gieson’s method

Collagen

§  Van gieson

§  Masson’s trichrome

§  Mallory’s aniline blue

§  Krajian’s aniline blue

Bone

·         Trichrome stains

·         PAS

·         Schmorl’s picrothionine method

·         Silver staining

·         Von kossa

·         Solochrome cyanine method

Neural tissue

·         Silver impregnation

·         Beilschowsky silver stain

·         PTAH

·         Cajal’s stain

Muscle

§  Modified Gomori’s trichrome stain

§  Rapid gomori trichrome

§  Mallory’s PTAH

§  Heidenhain’s iron hematoxylin

Elastic fibres

§  Weigert’s

§  Verhoeff’s

§  Gomori’s aldehyde fuchin

§  Krajan’s method

Plasma cells

§  Methyl green pyronine

Amyloid

·         Congo red

·         Sirius red

·         Toluidine blue

·         Crystal violet

·         Thioflavin T

Mast cell granules

·         Toluidine blue

·         Azure A

·         PAS

·         Alcian blue safranin

·         Keratin/keratohyaline granules

·         Performic acid alcian blue

Acidic stains	Basic stains
Eosin Erythrosine Fluoroscein Picric acid Alizarin Acid fuschin Bismark brown	Hematoxyline Acridine red Aniline blue Azure Basic fischin Crystal violet Malachite green Safranine

Vital stains ------ which do not kill the cells

Supravital staining----for live organisms, cells retain vitality after staining

Supravital stains-------

·         alizarin red--------- for developing bone

·         toludine blue---------- for precancerous lesions in oral cavity

Intravital staining---------- cells removed from body &  loose vitality after staining

Intravital stains----------India ink preparation