Cytochemical-Staining-Techniques (1).pdf

Section 1A & 1B  Storage form of glucose Normally stored in the liver, kidneys, and skeletal muscle. PERIODI Most versatile and widely C used for demonstrations of carbohydrates or glycoconjugates. ACID Stains polysaccharides, neutral...

Section 1A & 1B  Storage form of glucose Normally stored in the liver, kidneys, and skeletal muscle. PERIODI Most versatile and widely C used for demonstrations of carbohydrates or glycoconjugates. ACID Stains polysaccharides, neutral mucins SCHIFF Periodic Acid Treatment Formation of Aldehydes: Periodic acid cleaves vicinal diols in carbohydrates, forming aldehyde groups at the sugar chain ends. Reaction with Schiff Reagent Aldehyde-Dye-Reaction: This reaction involves the nucleophilic attack of the nitrogen in the Schiff reagent on the electrophilic carbon of the aldehyde, leading to the formation of a colored product. Color Development: Magenta  Nuclei - blue Polysaccharides, neutral mucins - red or magenta PAS 1. Mechanism of Action 2. Alternatives to Malt Diastase Alpha-amylase: Human Saliva: ⚬ Catalyzes the hydrolysis of glycosidic ⚬ Contains α-amylase but discouraged due bonds in glycogen. to safety concerns and lack of ⚬ Breaks down large glycogen molecules standardization. into maltose, a water-soluble disaccharide. ⚬ Net result is the removal of glycogen from the tissue section prior to PAS technique Malt Diastase: ⚬ Contains both α-amylase and β-amylase for efficient glycogen breakdown.  PAS PAS w/ Diastase Nuclei - blue Polysaccharides, neutral mucins - red or magenta  4. Tumor Analysis Carcinoid Tumors and Other Neoplasms 5. Diagnosis of 1. Glycogen Detection Inflammatory Conditions Diabetes Mellitus Chronic Inflammation Liver Pathology 6. Basement Membrane 2. Mucopolysaccharide Assessment Detection Renal Pathology Mucopolysaccharidoses 7. Identifying Certain Viral 3. Fungal Infections Infections Fungi Identification Cytomegalovirus (CMV)  Duration Exceptions Intensity of stain is Glutaraldehyde dependent fixative should be on the length of avoided if stained with treatment with the the PAS Periodic acid technique and Schiff reagent  Carleton’s Modification Glycogen stain Obsolete method. Rapid staining, but not permanent. Glycogen: Mahogany brown Tissue consituents: Yellow  Good staining technique for glycogen demonstration. ⚬ Affinity of alkaline carminic acid for glycogen Selective, but not specific. ⚬ Mast cell granules, fibrin, and mucin. Categorized as a derivative of a natural dye. + +  Nuclei: Blue or Grayish blue Glycogen: Pink to red (Bright Red) Mucin: Weak red  Polysaccharides bound to other substances Makes up ground substance of connective tissues Acid Mucins comprise the intercellular or ground substance throughout the body PAS (+/-), Alcian Blue (+), Colloidal Iron (+), and Metachromatic Stain (+) Neutral Mucins less distributed and less important than acid mucopolysaccharides found in epithelial and intestinal glands  Initially used for the dyeing of textile fiber. Composed of a central copper-containing phthalo- cyanine ring linked to four isothiouronium groups via thioether bonds. Different alcian blue dyes have been produced; Alcian blue 8GX is the recommended dye for histological techniques. Used to stain proteoglycan/hyaluronic acid components of connective tissue and cartilage. The isothiouronium Alcian blue then gives component of the dye The counterstain, connective tissues gives the stain a nuclear fast red, pH level must be and cartilages a blue positive charge, gives contrast to kept within 1.0 to color due to the allowing formation of other components of 2.5 to prevent abundance of electrostatic bonds the tissue that has deprotonation hyaluronic acid in with negatively no acidic these structures, as charged tissues components long as the pH is ~2.5 containing carboxyl or sulfate groups  Alcian Blue Nuclei - red Acid mucins - blue LOW PH ALCIAN BLUE TECHNIQUE COMBINED ALCIAN BLUE-PAS Useful in the Used to differentiate neutral characterization of the mucins from acidic mucins subtypes of acidic mucins within a tissue section and proteoglycans  Alcian blue and PAS Usually, sections are stained techniques can be combined with a standard Alcian blue to differentiate neutral method (pH 2.5) then mucins from acidic ones followed by PAS. within a tissue section. Tissues and cells that contain The alcian blue will stain both neutral and acidic sialomucins, sulfomucins mucins will stain varying and proteoglycans in blue. shades of purple due to the Neutral mucins will appear binding of alcian blue and the deep red/magenta in color reactivity with the Schiff with the PAS.  Alcian Blue Alcian Blue/PAS Nuclei - red Nuclei - pale blue Acid mucins - blue Acid mucins - blue Glycogen, Neutral mucins - red or magenta  A phenomenon where in basic dyes belonging to aniline group that will differentiate particular tissue components by giving them a different color to that of original dye. The tissue element change in color due to polymerization. The original color of the dye changes to another color (i.e. mast cell stained pink with toluidine blue) Identify charged mucins and proteoglycans One of the oldest histochemical techniques for carbohydrates  Reagent: Alcoholic azure A solution ⚬ Azure A - 0.01g ⚬ Ethanol 30% - 100mL Principle: Amyloid (a glycoprotein) exhibits methachromasia in tissue sections when stained with crystal violet and other cationic dyes Results: ⚬ Acid mucins and proteoglycans - purple Acid mucins and proteoglycans in purple to red color to red Background tissue in blue color ⚬ Other tissues - blue  On bonds: Metachromasia: Metachromasia is Carbohydrate: Staining solutions There is a shift in the believed to for dye The anionic Methylene blue, azure absorption of light by aggregation carbohydrates A and toluidine blue the tissue dye characterized by structure serves as a are small cationic complex towards the formation of new template to induce dyes - typycally stains shorter wavelengths intermolecular bonds formation of polymeric blue but stain purple- with an inverse shift in between adjacent dye dye structures. red in metachromasia. color transmission. molecules.  3. Evaluation of Myelin Sheaths: Nervous System Studies: Metachromatic staining helps visualize myelin in nerve tissues, allowing for the assessment of myelin sheath integrity in conditions like multiple sclerosis. 1.Diagnosis of Metachromatic Granules: 4.Assessment of Glycosaminoglycans: Corynebacterium diptheriae: The Mucopolysaccharides: Toluidine blue stains metachromatic granules (volutin granule) can can detect mucopolysaccharides - for be identified using methylene blue or albert detection of mucopolysaccharidoses. stain, aiding in the diagnosis of diptheria. 5. Histopathological Studies: Tumor Diagnosis: Metachromatic staining can 2.Detection of Mast Cells help differentiate between various types of Histamine and Heparin: mast cells contain tumors based on the presence of specific metachromatic granules rich in heparin and metachromatic substances. histamine. Aiding in allergy and asthma 6.Microbial Identification research Staining of Bacteria: certain bacterial specie exhibit metachromasia.  Aldehyde fuchsin was introduced as an elastic tissue stain by Gomori in 1950. Composition: Basic fuchsin + aldehyde Demonstrate sulfur-containing compounds. Basic fuchsin + acid-alcohol and paraldehyde mixture= acetaldehyde In the presence of strong mineral acid : basic fuchsin + certain aldehydes = Intensely purplish dyes  NOT soluble in water 2 to 3 days @ RT until sufficient amount of deep purple aldehyde- fuchsin is formed in the mixed solution In the presence of acid catalyst, paraldehyde depolymerizes gradually to give acetaldehyde which reacts with the open amino groups of basic fuchsin. Dye mixture should be made up two or three days in advance of each experiment USED TO STAIN: RESULTS OF STAINING: Acid mucopoly-saccharides Aldehyde fuchsin:⬆affinity for Sulfated mucosubstances sulfated mucins (stained purple) Pancreatic islets of Langerhans Carboxylate forms: sialomucins, e.g. Thyrotrophic hormones submandibular salivary gland and Secretory substances intestinal goblet cell (stained blue after Stain mast cells (w/o counterstaining) subsequent counterstaining with alcian blue)  EXPECTED RESULTS: RESULTS OF STAINING: Aldehyde fuchsin:⬆affinity for Elastic fibres – purple sulfated mucins (stained purple) Mast cells – purple Carboxylate forms: sialomucins, e.g. Hyaline Cartilage - purple submandibular salivary gland and Pituitary β cells – purple intestinal goblet cell (stained blue after subsequent counterstaining with alcian Sulphated mucins – purple blue) B-cells - Purple Background – as the counterstain Nuclei – as the nuclear stain Modified aldehyde fuchsin stain (Halmi's modification of Gomori's method) COUNTER STAIN: light green or orange G NUCLEI: Celestine blue or haemalum (alum hematoxylin) ORGANELLES: yellow, purple, or green COLLAGEN and the BASEMENT MEMBRANE: Green MAST CELLS, ELASTIC CELLS, AND MUCUS: purple  Aldehyde fuchsin stain showing loss of elaunin and oxytalan fibers in the papillary dermis with short fragmented elastic fibers in the reticular dermis. Pituitary Gland Mucin in the stomach The mucicarmine technique, an early histochemical method for observing mucins, has slightly decreased over the past 50 years due to alternative methods like PAS, colloidal iron, and alcian blue. It is useful for illustrating acidic mucins and identifying adenocarcinomas, particularly gastrointestinal tract adenocarcinomas. Carmine-Mucin Interaction: Mucicarmine solution pH Sensitivity: Selective Staining: Counterstaining: contains carmine, an acidic The mucicarmine dye The mucicarmine does not stain all Often, mucicarmine dye that is typically linked with aluminum salts to stain works carbohydrates; it staining is used in enhance its specificity for optimally in acidic specifically binds to acidic combination with acidic substances. conditions. At lower mucins such as those hematoxylin or The aluminum-carmine pH, mucins have a found in the extracellular other nuclear stains complex binds selectively matrix and some stronger negative to visualize cell to acidic glandular secretions (e.g., mucopolysaccharides charge, which in goblet cells or certain nuclei, as (mucins) found in the enhances the cancer cells, like mucicarmine tissue. These mucins are binding of the adenocarcinomas). primarily highlights carbohydrate molecules aluminum-carmine Other tissue components mucins in a red or with acidic groups (e.g., remain unstained or are complex. pink color while sulfate or carboxyl groups), counterstained by other which are the target of the nuclei appear blue. dyes in the staining stain. procedure. Purpose: High Iron Diamine-Alcian Blue (HID-AB) Staining Kit is used to distinguish between sulfated acid mucins and salivary acid mucins. Principle: N,N-dimethyl-phenylenediamine dihydrochloride and N,N-dimethyl-p-phenylenediamine dihydrochloride are both ammonium salts that have a positive charge when dissociated. The diammonium salt is combined with sulfated acid mucins to form a complex, which can be observed through a color change. The reaction is slow, and Ferric chloride is used as a catalyst to speed up staining. On one hand, the oxidation of the two ammonium salts forms a brown- black cationic chromogen, which speeds up staining. On the other hand, the pH of the stain solution decreases to 1.4. At this pH value, only the sulfate group reacts with the diammonium salt to form a complex of purple-brown to brown-black, as the carboxyl group on the section cannot combine with the diammonium salt. Subsequently, Alicin blue (pH 2.5) stains the carboxylated sialic acid mucus as blue, allowing the acidic mucus of the two main groups to be shown separately. Sulfated mucins and black- proteoglycans brown Sialomucins and hyaluronic blue acid   Positive Staining: Acidic carbohydrates, such as sulfated mucins, exhibit a blue color due to the formation of Prussian blue after potassium ferrocyanide treatment. Differentiation: Acidic mucins stain blue, while neutral mucins may remain unstained or pale. Localization: The intensity and distribution of the blue staining indicate the presence and location of acidic carbohydrates within the tissue. Diagnostic Utility: This technique helps diagnose conditions like tumors and mucin- secreting disorders based on the presence of specific carbohydrate structures.  “Acridine orange is a carcinogen when absorbed through the skin. Wear gloves when working with this stain.”  Acridine orange is a fluorescent stain (fluorochrome) Used to demonstrate acid mucins Mucin+ iron hematoxylin and acridine orange = selective brilliant orange fluorescence **Other fluorescence in the tissue are subdued by iron hematoxylin = black color Fixation: Formalin and other fixatives, except heavy metals. Sections: Frozen or paraffin Sections Disadvantage: ⚬ temporary and will only last for about 2 hours once the section is mounted. Limitations: ⚬ Cellular debris within a sample such as white blood cells, epithelial cells, and dead bacteria may distort the microscopic image. ⚬ Acridine orange is a very sensitive stain, and caution should be used when interpreting results.  Place in 4% aqueous Wash briefly in Sections to water iron hemalum for 5- running tap water. 10 minutes. Stain in 0.1% aqueous Examine immediately Wash briefly and acridine orange for in a fluorescence mount in glycerin. 1% minutes. microscope.  Acid Background Fungi mucopolysaccharides - - - Reddish orange Greenish red Black fluorescence flourescence  Acridine orange stain can also aid in detecting Acanthamoeba infections, infectious keratitis, Helicobacter pylori gastritis, and cell wall deficient bacteria such as Mycoplasma. Enumerating the microbial load in a sample since acridine orange binds with the nucleic acid of living and dead bacteria. Detecting cell wall-deficient bacteria (e.g., mycoplasmas) grown in cultures. Cell wall- deficient bacteria are difficult to visualize in Gram stain as they cannot retain Gram stain dyes. Differentially staining human cells and prokaryotic cells with a fluorescence microscope. Human cells are stained black to faint green, making bright orange organisms easily detectable. Utilizing acridine orange in a method called quantitative buffy coat (QBC), a rapid screening tool for the detection of malaria. Analyzing mitochondria and lysosomal content by flow cytometry. Visually detecting nucleic acids on agarose and polyacrylamide gels. Identifying engulfed apoptotic cells because they will fluoresce upon engulfment.