Histology Staining Techniques PDF

PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING NATURAL DYES NATURAL DYES HEMATOXYLIN OUTLINE ● • • • Staining STAINING Is the process of applying dyes on the sections to see and study the architectural patterns of the tissue and physical characteristics of the cells. Tissue com...

PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING NATURAL DYES NATURAL DYES HEMATOXYLIN OUTLINE ● • • • Staining STAINING Is the process of applying dyes on the sections to see and study the architectural patterns of the tissue and physical characteristics of the cells. Tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution A colored compound is used to produce a contrast between different tissues and cellular components based on their varying affinities for most dyes and stains. PURPOSE OF STAINING • • Better microscopic visualization Differentiation – to identify tissue and cell compartments o To check for abnormalities in malignant cells, as well as infections or inflammations • Contrast (background) 1. Histological Staining tissue constituents are demonstrated in sections by direct interaction with dye - also called as Micro-anatomical staining - Micro-anatomic stains, bacterial stains and specific tissue stains (e.g. muscles, connective tissue and neurologic stains) fall into this category. 2. Histochemical Staining - various constituents of the tissues are studied through chemical reactions 3. Immunohistochemical - combination of immunologic and Staining histochemical techniques • Histologic Stain - Purified form of a coloring agent or crude dye that is generally applied in an aqueous solution. COCHINEAL DYES AND ITS DERIVATIVES ORCEIN SAFFRON SOURCE Logwood (Hematoxylin campechianum) Female Cochineal Bugs (Coccus cacti) Lichen (Roccella tinctoria) Saffron (Crocus sativus) SYNTHETIC DYES PROPERTIES OF DYES • • • • • • CHROMOSPHORE – the coloring property AUXOCHROME – the dyeing property HEMATOXYLIN Staining solution most commonly used for routine histologic studies Active coloring agent is HEMATIN – formed by the oxidation of Hematoxylin in a process called “RIPENING” (natural and artificial) Ripening/oxidation of hematoxylin is done by: o Exposing to sunlight and air (slow) o And adding strong agents: ▪ hydrogen peroxide ▪ mercuric oxide ▪ potassium permanganate ▪ sodium perborate ▪ sodium iodate Ripened Hematoxylin – rarely used due to low affinity to tissue. NOTE: The great majority of routine histology is done with hematoxylin and eosin (H&E) staining, because it is quick, cheap and informative. It involves the use of two contrasting stains. Hematoxylin which stains the nuclear detail, and Eosin which brings out the cytoplasmic detail of the cell and the tissue's architecture. TYPES OF STAIN (According to Source) NATURAL DYES Hematoxylin Cochineal Dyes Orcein Saffron SYNTHETIC (ARTIFICIAL DYES) Aniline Coal Tar Dyes TYPES OF STAIN (According to Effect) ADJECTIVE SUBSTANSIVE AMPHOTERIC Acts on tissue but first treated with another substance Acts immediately, directly on the object Acid and basic grouping TYPES OF HEMATOXYLIN 1. 2. 3. 4. 5. 6. Aluminum hematoxylin Iron hematoxylin Tungsten hematoxylin Molybdenum hematoxylin Lead hematoxylin Hematoxylin without mordants MORDANTS • tissue and staining ; still remain Substances that combine with the tissue and the staining solution, forming a bridge that allows staining reaction to take place. o When excess dye solution is washed away, the mordanted stain remains. o Many dyes require a mordant Last Name PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING RIPENING AGENTS HEMATOXYLIN Weigert’s Hematoxylin Heidenhain’s Hematoxylin Erlich’s Hematoxylin Mayer’s Hematoxylin Harris Hematoxylin Cole’s Hematoxylin NOTE: The difference between the mordant and the accentuator is that the accentuator is not part of the chemical reaction, it only helps speed up the staining of the tissue, while the mordant allows the reaction of the stain to reach the tissue. • • MORDANTS - integral part of the staining reaction, serves as a link or bridge between the tissue and the dye ACCENTUATORS - not essential to the chemical union of the tissue and the dye. It does not participate in the staining reaction, but merely accelerates the reaction cochi bug • • • • ALUMINUM HEMATOXYLIN • Active mordant: Alum/Potassium Aluminum Sulfate HEMATOXYLIN Erlich’s Hematoxylin Harris Hematoxylin COMPONENTS Hematoxylin, Absolute ETOH, Alum, Glycerin, Dist. H2O, GAc Hematoxylin, Absolute ETOH, Alum, Dist. H2O, Mercuric Chloride, GAc Cole’s Hematoxylin USE Regressive staining • • • • Widely used for routine nuclear staining in Exfoliative Cytology and sex chromosomes Recommended for routine purposes Hematoxylin, 1% Iodine in 95% ETOH, Sat Aq Ammonium Alum, Dist H2O Mayer’s Hematoxylin, Used in Celestine Hematoxylin NaIO3, Alum. Citric Blue Hemalum Acid, Chloral Method of nuclear Hydrate Dist H2O staining Others: Lillie’s Hematoxylin, Delafield Hematoxylin • • • • • Heidenham’s Hematoxylin Phosphotungstic Acid Hematoxylin (PTAH) COMPONENTS Hematoxylin, Absolute ETOH, FeCl, HCl, Dist H2O Hematoxylin, 95% ETOH, Fe Ammonium Sulfate, Dist H2O Hematoxylin, Phosphotungstic Acid, Dist H2O USE For demonstration of muscle fibers and connective tissues Regressive staining of thin sections: staining of voluntary muscle striations and myelin Demonstration of structures in paraffin, celloidin, frozen sections → ORCEIN Vegetable dye from Lichens Colorless and if treated with ammonia and expoed to air produces blue or violet colors For elastic fibers Litmus – used as indicator EOSIN Counterstain after hematoxylin and before methylene blue 2 shades: Bloody Red o Bluish (Eosin B) – deeper red color o Yellowish (Eosin Y) – most commonly used; in both aqueous and alcoholic solutions (yellow fluorescence) Eosin Y –0 5g Distilled H2O – 100mL Thymol EOSIN (ALCOHOLIC SOLUTION) • • • • Active mordant: Iron Alum/Ferric Aluminum Sulfate HEMATOXYLIN Weigert’s Hematoxylin COCHINEAL DYES From female cochineal bug (Coccus cacti) + alum carmine Powerful chromatin and nuclear stain With picric acid – picrocarmine – neuropath With AlCl3 (Best’s Carmine) – glycogen 5% AQUEOUS EOSIN Y 0 IRON HEMATOXYLIN • RIPENING AGENTS Ferric Chloride Ferric Ammonium Sulfate Sodium Iodate Sodium Iodate Mercuric Oxide Alcoholic Iodine Solution FOCUS Eosin Y – 1g Distilled H2O – 20mL 95% alcohol – 80mL Gac (optional) – 0.5mL to give a deeper red stain 0 soon ! METHYLENE BLUE METHYLENE VIOLET TOLUIDINE BLUE lpnystal CRYSTAL VIOLET protein Foton section and platelets in Blood ANILINE BLUE BASIC FUCHSIN Basil SMAC ! ! OTHER STAINS Basic nuclear stain with eosin; for plasma cells; sputum, bacterial stain, vial staining of nervous tissue, indicator Metachromatic dye Nucelar stain – Nissl granules, chromophilic bodies used ! ! Nucelar stain or chromatin stain used for staining amyloid in frozen section and platelets in blood. Crystal violet if with methyl violet & dextrin → Gentian violet Cytoplasmic counterstain Acid fast, mitochondria, smooth muscles Last Name PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING • • • • SMAC VAN GIESON’S 101191 Poll a) = Pink = Black ) flag 1 Batak MASSON’S TRICHROME STAIN GIEMSA CELESTINE BLUE MALACHITE GREEN METHYL GREEN BISMARCK BROWN PRUSSIAN BLUE ORCEIN PICRIC ACID CARMINE + AlCl3 MAYER’S CARMALUM SOLUTION IODINE Carbol fuschin Schiff’s reagent Mallory’s Fuchsin Aldehyde Fuchsin (Gomori’s stain) Picric acid + acid fuschin Collagen → pink Elastic fiber → black For connective tissues É Blood Good nuclear stain with Alum hematoxylin Ascaris ova, RBC, spore, decolorizer, counterstain Chromatin Contrast, diphtheria Use to manufacture paint Elastic fibers – Taenzer Unna; dermatologic studies – fibers Contrast stain to acid fuchsin – demonstration of CT; counterstain to crystal violet, tissue fixative, decacifier Glycogen – best carmine and fuchsin (Mucicarmine) Basic dye and stains acidic structures : • Sudan GOLD SUBLIMATE Used for Metallic impregnation – made up of gold chloride (brown) and mercuric chloride o Mercuric chloride – 0.4g o Distilled H2O – 60mL o 1% gold chloride – 10mL SILVER NITRATE Stains spirochete, reticulum, other fiber stains o Silver Nitrate solution – 10mL o Distilled H2O – 90mL • • • • • CHIEF SOLVENTS USED FOR STAINS Water – always used unless otherwise stated Alcohol – ETOH, MTOH; should be acetone-free Aniline water Phenol METHODS OF STAINING DIRECT VS. INDIRECT • • Direct staining – sections are stained with simple of aqueous solution of the dye o giving color to the sections by using aqueous or alcoholic dye solutions. o Only one dye is used, which is washed away after 30–60 seconds, prior to drying and examination. Indirect staining – requires mordant and accentuator for staining o Action of the dye is intensified by adding another agent/mordant o "Tissue-mordant-dye-complex" mordant combines with a dye to form a colored "lake", which in turn combines with the tissue. o Rendered insoluble in ordinary aqueous and alcoholic solvents. o Allows subsequent counterstaining and dehydration to be carried out easily PROGRESSIVE VS. REGRESSIVE • • Black OIL SOLUBLE DYES – LYSOCHROMES Sudan to "I • No auxochrome Sudan • Gives color to lipids because they are more soluble in lipid medium of the tissues, than in their medium of 70% alcohol • Sudan Black, Sudan IV (Scharlach R), Sudan III OSMIUM TETROXIDE Fixative and also stains fat AMMONIA WATER Used to blue stains o Strong ammonium hydroxide – 2mL o Tap water – 98mL NOTE: also referred to as “the bluing step” • Oldest, 0 starch, amyloid, corpora amylacea ALCIAN BLUE Phthalocyanin dye similar to chlorophyll - stains acid MPS - more specific for CT and epithelial mucin NEUTRAL RED Basic dye – cell granules CONGO RED Indicator – stain for axis cylinders in embryos – Krajan’s method – elastic Janus need Power tissues, amyloid, myelin JANUS GREEN B Mitochondria (intravital) VICTORIA BLUE I Neuroglia (FS) NIGHT BLUE Night FulkCarbol-fuchsin – acid fast ACRIDINE RED 3B Calcium deposits ACRIDINE ORANGE Green fluorescence for DNA and red if RNA RHODAMINE B w/ osmic acid-blood/glands BENZIDINE Hemoglobin • • Progressive staining – tissues are stained in a definite sequence Regressive staining – tissues are first overstained & excess stain is removed or decolorized METACHROMATIC • Metachromatic staining – stain imparts a different color to the tissue o Example: Toluidine Blue impparts red-purple to Mast cells COUNTERSTAINING • Counterstain staining – application of a different stain to provide contrast and background to the staining of the structural components to be demonstrated Last Name PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING o EX. Eosin and Safranin MICROANATOMICAL • nucleus MCLSBBNITTA w/o inclusion Bodies Microanatomical staining – demonstrate the general relationship of tissues with general differentiation of nucleus and cytoplasm without necessarily emphasizing the inclusion bodies o Cytoplasmic staining o Negative staining METALLIC IMPREGNATION • Metallic impregnation – specific tissue elements are demonstrated by colorless solutions of metallic salts which are reduced by tissues producing an opaque black deposit on the surface of the tissue • • • VITAL – INTRAVITAL VS. SUPRAVITAL • intra • - galoob • • • • • • • Vital staining – stain that can ve applied on living cells without killing them Intravital staining – non-toxic dye injected into the body to selectively stain certain cells or tissues Supravital staining – stain living cells immediately after removal from the body o Ex. Crystal violet, bromocresol blue, and new methylene blue Neutral red (the best vital dye) Janus green (mitochondria) Trypan blue Nile blue Thionine Toluidine blue INTRAVITAL STAINS India Leemak car SUPRAVITAL STAINS SUPRA JMT titi ni Red NUCLEAR STAINS Busy c. Med tech India ink Lithium Carmine Neutral red Janus green ITrypan blue Nile blue Tionine Toluidine blue Neutral red Safranin O Carmine Hematoxylin Methylene blue Toluidine blue Celestine blue Eosin Y Eosin B Phloxine B Picric acid Orange G Rose bengal Light green SF Lissamine green Methyl violet Crystal violet JR - PEEL PORI METACHROMATIC STAINS • • - - CYTOPLASMIC STAINS • ] .csaNa Hay • • Busy l medtelh Cresyl blue Safranin Basic Fuchsin Bismarck brown Methylene blue Thionine Toluidine blue Azure A,B,C STAINING OF PARAFFIN SECTIONS H&E STAINING PROCEDURE Paraffin wax is poorly permeable to most stains o removed from the section prior to staining. Immerse paraffin section in a solvent (e.g. xylene) 2 times, at 1-2 minutes duration each, for sections up to 10 micron thick. o Xylene - not miscible with aqueous solutions and low graded alcohol, and is removed with absolute alcohol, o “Sections to Alcohol” - Descending grades of alcohol to prevent damage and detachment of sections. o “Sections to Water” - If tissue has been fixed in mercuric chloride Alcohol is replaced with water before staining of sections o If an alcoholic stain is to be used, there is no more need to replace the alcohol After the section is cut and mounted on the slide, it is drained and dried o If drying is not complete, the section (or part of it), especially from bone and nervous tissue, may become detached from the slide during the process of staining After staining, the section is again dehydrated with increasing grades of alcohol and cleared with two changes of xylene to prepare the section for mounting, o Most mountants are miscible in0 xylene - second change of xylene raises refractive index of the glass slide o reduces light refraction during microscopic examination. The section should not be allowed to stay in alcohol for a long time because many stains are usually removed by prolonged immersion in alcohol. Sections must be left in the oven for a minimum of0 30 minutes before they are finally stained. o Slide is dirty or greasy - May float off the slide during staining REAGENT Xylene (2 changes) Descending grades of ROH Hematoxylin 1% acid alcohol Ammonia water Eosin Ascending grades of ROH Xylene (2 changes) PURPOSE Deparaffinization + clearing Hydration of alcohol Primary stain Differentiation Blueing agent Counterstain Dehydration of alcohol Clearing Last Name PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING MATERIALS FOR STAINING COPLIN JAR SLIDE DISH WITH STAINING RACK/CENTER • • • • • • • • RESTAINING Xylene – for 24 hours Coverslip is removed using dissecting needle Xyleen – 30 minutes 0.5% Potassium Permanganate – 5-10 minutes Rinse with tap water 5% Oxalic acid – 5 minutes Rinse with tap water Allow to dry and restain METAL OF GLASS STAINING RACK/CARRIER SLOTTED STAINING DISH WATCH GLASS SECTION LIFTER PIPETTE SCREW CAP VIAL STAINING RACK Last Name PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING STAINS AND STAINING SOLUTIONS STAINS OF CARBOHYDRATES PERIODIC ACID SCHIFF (PAS) PAS WITH DIASTASE BEST CARMINE LANGHAN’S IODINE " " "" ^ FRESH FROZEN AZURE A [ METACHROMATIC TOLUIDINE BLUE STAINING ALCIAN BLUE TECHNIQUE ALCIAN BLUE AI Clan asana ni Scott's Mowry 's COMBINED ALCIAN BLUEPAS TECHNIQUE MUCICARMINE STAIN HALE’S DIALYZED IRON TECHNIQUE FLUORESCENT ACRIDINE ORANGE Result: PAS (+) – red or magenta Nuclei – blue - Method of choice for glycogen demonstration Result: Nuclei – blue-black Gycogen – red Control – only the nuclei are stained - For glycogen demonstration Result: Nuclei – blue or grayish-blue (Erlich’s hematoxylin as counterstain) Glycogen – bright red granules Mucin, fibrin – weak red - Oldest stain (obsolete) Result: Glycogen – mahogany brown Tissue constituents – yellow - Metachromatic staining for glycoaminoglycans Result: Glycosaminoglycans – redpurple Tissue background – blue Result: Glycosaminoglycans – redpurple Tissue background – blue Result: Acid mucin – blue Nuclei – red - Used primarily for demonstrating acid mucopolysaccarides with scott’s method and mowry’s staining methods - used in electrophoresis for detecting glycoproteins - For acid and neutral mucin Result: Acid mucin – blue Neutral mucin – magenta Nuclei – pale blue Result: Mucins – red Nuclei – blue Backgroud – unstained Result: Acid mucin – dark blue Nuclei – red Result: Acid mucopolysaccharides – black Fungi – greenish red fluorescent Background – reddish orange fluorescent - DNA intercalating dye - A grade of acridine orange of exceptionally high purity, suitable for quantitative work. Free of inorganic salts - A specific stain for RNA, used as a 2% solution containing 1% lanthanum acetate in 15% acetic acid ACRIDINE ORANGE 0 STAINS OF FATS SUDAN BLACK B - SUDAN IV (SCHARLACH R) - OIL RED O IN DEXTRIN - OSMIC ACID : OIL RED O - Most sensitive Result: Lipids - blue black Nuclei - red Result: Lipids (mainly triglycerides) - red Nuclei – blue or black Result: Fats – brilliant red Nuclei – blue Result: fats – black Nuclei – yellow orange - Excellent for the demonstration of the general localization of fats in frozen tissue sections STAINS OF PROTEIN, ENZYMES AND NUCLEIC ACID ALKALINE FASTGREEN GOMORI CALCIUM FEULGEN METHYL PYRONIN GREEN- FAST GREEN FCF Result: Histones and protamines – green Result: Alkaline phosphatase activity – brownish black Nuclei – green - For nuclear DNA Result: DNA – red-purple Cytoplasm – green - For RNA and DNA Result: DNA – green or blue-green RNA – rose red Plasma cell cytoplasm – purple - Sensitive stain for proteins in polyacrylamide gels - especially suitable in isoelectric focusing - also suitable for use as a cytological counterstain, and mammalian tissue stain for muscle and collagen Last Name PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING STAINS OF CONNECTIVE TISSUE STAINS OF BONE MARROW AND BLOOD ELEMENTS GOMORI’S SILVER IMPREGNATION STAIN - For reticulin Result: Reticulin fibers – black VAN GIESON’S STAIN - For collagen Result: Collagen (fibrous connective tissue) – pink, deep red Nuclei – brownish black Muscle, cytoplasm, RBC and fibrin – yellow MASSON’S TRICHROME - For collagen STAIN Result: Collagen and mucus – blue Muscle, RBC and keratin – red Nuclei – blue-black Other stains for collagen: Mallory’s aniline blue, Azocarmine, Krajian’s Aniline Blue lmak ) MASSON’S TRICHROME - Used for the detection of collagen STAIN KIT fibers in tissues such as skin, heart, etc. on formalin-fixed, paraffin-embedded sections and may be used for frozen sections as well. - collagen fibers stained blue - nuclei stained black - cytoplasm, muscle, erythrocytes stained red FOR ELASTIC FIBERS Weigert’s Elastic Tissue Stain • Result: Elastic fiber – dark blue, blue black Verhoeff’s • Result: Elastic fibers – black WVTGK Nuclei – gray to black Collagen – red Cytoplasm – yellow Taenzer-Unna Orcein Gomori’s Aldehyde-Fuschin Krajian’s FOR FIBRIN Mallory’s PTAH FOR AMYLOID GINO Amy ! ! Gram’s iodine Congo red Methyl violet – crystal violet STAINS OF MUSCLES AND BONES MODIFIED GOMORI’S TRICHROME STAIN Rapid Toluidine-Eosin Stain RRWGP Romanowsky Wright’s stain Giemsa stain Peroxidase reaction – for myeloid cells STAINS OF CNS BIELSCHOWSKY’S TECHNIQUE BIELSNANI technique For neurons, axons, and neurofibrils BODIAN’S STAIN Bodian 's Meng Nerve fibers and nerve endings SIEVER-MUNGER TECHNIQUE Neural tissues CRESYL FAST VIOLET Nissl bodies in neurons and cell nuclei - - - THE BIELSCHOWSKY STAIN - Very useful method in detecting the twist and clumping of neruons in the brain, otherwise known as neurofibrillary tangles and senile plaques, (signs of the Alzheimer’s disease). - the Bielschowsky stain, along with other diagnostic test, can assist scientist and researchers in the mission for finding a cure for this disease of memory loss. FOR MYELIN SHEATH WKW Weigert-Pal Technique Kluver and Barrera Luxol Fast Blue Stain Weil’s Method FOR ASTROCYTES Cajal’s Gold Sublimate Modified PTAH Modified Holzer’s Method Result: Muscle fiber – red Collagen – green Nuclei – blue to black Mallory’s PTAH Heidenhain’s Iron Hematoxylin Lissamine Fast Red – tartrazine method for muscles and bones FOR BONES Schmorl’s Picro-Thionin Method Last Name PRELIMS | HISTOPATHO | SECTION Week 10 | Prof. Caccam | STAINING STAINS OF TISSUE PIGMENTS AND DEPOSITS FOR HEMOSIDERIN Perl’s Prussian Blue Gomori’s Prussian Blue Turnbull’s Blue Reaction FOR HEMOGLOBIN Benzidine-Nitroprusside Stain FOR BILE PIGMENTS AND HEMATOIDIN Modified Fouchet’s Technique Gmelin’s Technique Stein’s Iodine PRUSSIAN BLUE OR PERL’S REACTION • Used to demonstrate ferric iron and ferritin • FIXATION: 10% NBF or alcohol can be used • Sections should be cut at 4μ to 5μ or blood or bone marrow smears. FOR FUNGI Grocott Methamine Silver FOR VIRUS Lendrum’s Phloxine-Tartrazine Method – viral inclusion Orcein Method – HbsAg FOR PROTOZOA Giemsa SUMMARY STAINING • • Natural dye Artificial dye Method of staining • Direct, indirect progressive, regressive, metachromatic, vital, intravital, supravital, counterstaining, microanatomical, metallic impregnation Different stains • According to usage and purposes FOR LIPOFUSCHIN Gomori’s Aldehyde Fuchsin Technique Mallory’s Fuchsin Stain FOR MELANIN Masson Fontana Technique – also for Argentaffin granules FOR CALCIUM Von Kossa’s Silver Nitrate method FOR COPPER Lindquist Modified Rhodanine Technique STAINS OF MICROORGANISMS FOR BACTERIA Gram’s method Brown and brenn – for Nocardia and Actinomyces Ziehl Neelsen – for Mycobacterium Wade-Fite Technique – M. leprae, Nocardia Auramine-Rhodamine – Mycobacteria Toluidine Blue and Cresyl Violet Actate – H. pylori Dieterle Method – L. pneumophilia Levaditi’s – Spirochete Modified Steiner and Steiner – Spirochete Warthin-Starry – Spirochete Last Name