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    HISTOCHEMISTRY

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    NAKUL
    Histochemistry combines histology and chemistry to reveal the biochemical composition of tissues through chemical or physical tests without disrupting the normal distribution. It aims to identify, localize, and quantify specific substances, reactive groups, and enzyme activities in cells and tissues. Common histochemical stains are used to demonstrate carbohydrates, nucleic acids, lipids, amyloid, microorganisms, connective tissue, and pigments. Periodic acid-Schiff stain detects glycogen and mucins. Congo red stain identifies amyloid deposits. Masson's trichrome stain colors collagen blue/green and cytoplasm/muscle red. Histochemistry is an important diagnostic tool in pathology.

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    HISTOCHEMISTRY

    Uploaded by

    NAKUL
    78 views63 pages
    Histochemistry combines histology and chemistry to reveal the biochemical composition of tissues through chemical or physical tests without disrupting the normal distribution. It aims to identify, localize, and quantify specific substances, reactive groups, and enzyme activities in cells and tissues. Common histochemical stains are used to demonstrate carbohydrates, nucleic acids, lipids, amyloid, microorganisms, connective tissue, and pigments. Periodic acid-Schiff stain detects glycogen and mucins. Congo red stain identifies amyloid deposits. Masson's trichrome stain colors collagen blue/green and cytoplasm/muscle red. Histochemistry is an important diagnostic tool in pathology.

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    Histochemistry combines histology and chemistry to reveal the biochemical composition of tissues through chemical or physical tests without disrupting the normal distribution. It aims to identify, localize, and quantify specific substances, reactive groups, and enzyme activities in cells and tissues. Common histochemical stains are used to demonstrate carbohydrates, nucleic acids, lipids, amyloid, microorganisms, connective tissue, and pigments. Periodic acid-Schiff stain detects glycogen and mucins. Congo red stain identifies amyloid deposits. Masson's trichrome stain colors collagen blue/green and cytoplasm/muscle red. Histochemistry is an important diagnostic tool in pathology.

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    78 views63 pages

    HISTOCHEMISTRY

    Uploaded by

    NAKUL
    Histochemistry combines histology and chemistry to reveal the biochemical composition of tissues through chemical or physical tests without disrupting the normal distribution. It aims to identify, localize, and quantify specific substances, reactive groups, and enzyme activities in cells and tissues. Common histochemical stains are used to demonstrate carbohydrates, nucleic acids, lipids, amyloid, microorganisms, connective tissue, and pigments. Periodic acid-Schiff stain detects glycogen and mucins. Congo red stain identifies amyloid deposits. Masson's trichrome stain colors collagen blue/green and cytoplasm/muscle red. Histochemistry is an important diagnostic tool in pathology.

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    of 63

    HISTOCHEMISTRY

    Vishnu K.
    TVM/20-02
    Dept. Of Vet. Pathology
    Histochemistry can be defined as 'the
    identification, localization and quantification,
    in cells and tissues and by chemical or
    physical tests, of specific substances, reactive
    groups and enzyme-catalysed activities
    Basic Principle

    Histochemistry combines the methods of


    histology with those of chemistry or
    biochemistry, to reveal the biochemical
    composition of tissues and cells beyond the
    acid-base distribution shown by standard
    staining methods (Hx & E), without disrupting
    the normal distribution of the chemicals.
     Goal of Histochemistry 
    • Presentation of Normal Chemical Distribution

    • Presentation of Normal Chemical


    Composition

    • Specificity of the Reaction

    • Detectability of the Reaction Product


    Applications
     Identify, quantify, and localize
    -Chemical substances
    -Gene expression
    -Biological structures, organelles
    -Specific cell types

     Clarify cell and tissue structure and morphology.


     Demarcate functional boundaries.
    Common terminologies in
    Histochemistry
    •  DYE: Coloured organic compounds that can
    be selectively bind to tissues.
    Eg: Benzene derivatives 

    • CHROMOPHORES: Any group that makes an


    organic compound coloured
    • AUXOCHROMES: Functional groups with non-
    bonded electrons which augments the
    colouring property of chromophores

    • MORDANTS: Non dyeing compound to


    improve binding of dye
    - Mediate a dye –tissue interaction
    - Greater stability to stain 
    • DYE LAKE: Dye - mordant complex

    • ACCELERATORS: To improve staining reactions

    • METACHROMASIA: Term used when dye


    stains a tissue component different color to
    the dye solution.

    • ORTHOCHROMASIA: Same color to the dye


    solution
    Special Histochemical stains for Routine
    diagnosis
    Special stains for Tissue Types

    •Carbohydrate
    •Nucleic Acid
    •Lipid
    • Amyloid
    • Stains for Micro-organisms
    •Connective Tissue Stains
    •Pigments and Minerals
    Carbohydrate Staining
     
    • Periodic acid schiff (PAS) technique
    • Alcian blue method
    • Combined alcian blue- PAS techhnique
    • Mucicarmine technique
    • Colloidal iron technique
    • Metachromatic staining
    • Iodine staining for glycogen
    • Enzymatic digestion technique
    -Diastase digestion
    - Sialidase digestion
    -Hyaluronidase digestion
    Periodic acid schiff method

    Glycogen and mucins : magenta Glutaraldehyde as


    Nuclei : blue fixative is avoided for
    PAS stain
    Periodic Acid Schiff (PAS) With
    Diastase
    PRINCIPLE - Glycogen is digested with certain
    forms of amylase.
    -Commercially available diastase or salivary
    amylase from saliva can be used to digest
    glycogen in tissue sections

    RESULTS- Presence of glycogen will be evidenced


    by loss of staining after enzyme treatment
    when compared to the untreated section
    Uses of PAS stain

    • Fungi: stains fungal cell walls


    • Kidney:
    -Routine evaluation of renal biopsies(basement
    membrane staining)
    -Renal cell carcinoma (stains glycogen, removed by
    diastase)
    • Liver:
    -Routine stain for hepatocytes (PAS without diastase)
    -Inclusions of alpha-1-antitrypsin disease
    • Lung: BAL fluid in pulmonary alveolar
    proteinosis
    • Muscle biopsies: routine stain to
    demonstrate glycogen
    • Pancreas: acinar cell carcinoma
    Alcian Blue
    • At pH 2.5 it stains – Acidic mucin that include
    acid-simple or non- sulfated and acid –simple
    mesenchymal mucin.
    • At pH 1 it stains acid-complex or sulfated
    mucin
    • At pH 0.5 it stains acid-complex connective
    tissue mucin.
    • It does not stain neutral mucin.
    Results
    • Sulfomucin
    • Sialomucin Blue
    • Proteoglycans
    • Hyaluronic acid

    • Nucleus - Red
    Combined Alcian Blue- PAS Technique

    Principle :
    • Demonstrate presence of mucin
    • Differentiate acid mucin from neutral mucin
    • 1st stain all acid mucin with alcian blue (blue)
    • Those acid mucin which are PAS +ve will not
    be stained on PAS reaction
    • Only neutral mucin will be stained(magenta)
    Mucicarmine
    • Carmine complex has a positive charge and so
    attracts polyanions such as sialomucins and
    sulfomucins

    • Useful for identification of adenocarcinoma


    (especially of GIT)

    • Capsule of fungus – Cryptococcus neoformans


    is also detected
    Results :
    • Acidic mucins – deep rose to red
    • Nuclei – black
    • Other tissue elements – light yellow
    Hale’s Colloidal Iron Stain
    Positive staining with hale’s colloidal iron stain is
    considered diagnostic feature of chromophobe renal
    cell carcionoma and has been used as discriminatory
    feature to differentiate it from other renal tumour.

    Result
    Acid mucopolysaccharides: blue
    Nuclei: red
    Nucleic acids
    • Demonstration of Nucleic acids depends upon
    either
    - Reaction of the dyes with the phosphate groups
    - Production of aldehydes from the sugar
    (deoxyribose)

    • No histochemical methods are available to


    demonstrate the nitrogenous base
    DNA Demonstration
    •  Feulgen technique ( demonstrate sugar)

    • Methyl green pyronin technique (demonstrate


    phosphate)

    • Acridine orange (by fluorescent method) – Both


    DNA & RNA

    • Gallocyanin-chrome alum method


    Feulgen Stain
    • DNA : red-purple
    • CYTOPLASM : green
    Methyl Green Pyronin Method
    • DNA : green-blue
    • RNA : red
    LIPID STAINS
    • Lipids with melting point below staining temperature
    can be stained with fat stains
    • only lipids which are liquid at staining temp. are
    stained.
    • Those in solid or crystalline state remains unaffected
    • Melting point of a lipid is inversely related to its fatty
    acid chain length
    • Simple lipid is best demonstrated with fresh frozen
    sections
    • Best fixative - Formal calcium (2% calcium acetate
    +10% formalin)
    Sudan Black B
    • Sudan Black B has 2 fractions –
    - 1st stains neutral fats blue-black
    - 2nd stains phospholipids gray

    • This gray reaction can be enhanced as a bronze


    dichroism if section is viewed in polarised light
    • It fails to stain crystalline cholesterol, lecithin and free
    fatty acids
    • Bromine pre treatment converts crystalline cholesterol
    to oily derivatives and hence permeable to Sudan dye
    Oil Red O
    • Principle :
    • Frozen or cryostat sections are used.
    • Staining with oil-soluble dyes is based on the greater
    solubility of the dye in the lipid substances
    • Inorder to penetrate fats,dye should be dissolved
    with organic solvents.
    • 70% ethanol – adequate solvent
    • Aqueous mounting medium - organic solvents found
    in synthetic resinous media will dissolve the fat.
    Disadvantages:
    • Fat is relatively liquid, so mounting should be done
    carefully.
    • Some neutral fat my be lost during staining
    • Technique sensitive
    Osmium Tetroxide for Unsaturated Lipids

    • Unsaturated lipids – brown to black


    • Saturated lipids /free cholesterol – do not
    react

    NB: Handled carefully as toxic vapour affect cornea


    and mucous membranes
    Amyloid
    • Stain used- Congo Red (gold standard), Sirius
    Red
    • Metachromatic Techniques - Crystal violet,
    Methyl violet, Methyl Green
    • Polarising Microscopy- Apple green
    birefringence
    Congo Red Stain
    • It stains amyloid by hydrogen bonding and
    other tissue components by electrochemical
    bond

    •  Amyloid - red
    • Nuclei – Blue
    Congo red staining shows amyloid deposition in renal biopsy
    Methyl /Crystal Violet Method 
    • Amyloid stains metachromatically (i.e., a
    different color from the dye solution, in this
    case red rather than violet) with crystal violet
    Stains for Microorganisms
    Gram Staining for Bacteria
    Gram positive bacteria – blue
    Gram negative bacteria – red
    Nuclei– red
    Other tissue elements - yellow
    Ziehl Neelson Acid Fast Staining

    Acid fast bacilli -bright red


    Other tissue -Pale blue
    Caseous material -very pale grayish blue 
    Warthin Starry Method for Spirochetes 

    SPIROCHETES – black
    BACKGROUND – golden -yellow
    Fungal Stains
    Gomori Methenamine Silver nitrate(GMS)
    technique
    Fungi , Pneumocystis, melanin - Black
    Mucin & Glycogen - dark grey
    Background - Pale green
    Hyphae & yeast form - sharply delineated in black
    against green background
    Connective Tissue Stains
    Collagen Fibres
    • Masson’s trichrome technique
    • Van Gieson’s stain
    • Mallory’s Phosphotungstic Acid Hematoxylin
    • PAS
    • Heidenhain’s Azan stain
    • Lillie’s allochrome method
    • Luxol fast blue G
    Masson’s Trichrome technique Principle:
    • The term ‘trichrome stain’ is a general name
    for a number of techniques for selectively
    demonstration of muscle, collagen fibers,
    fibrin, and erythrocytes.
    • The general rule in trichrome staining is that
    the less porous tissues are colored by the
    smallest dye molecule; whenever a dye of
    large molecular size is able to penetrate, it will
    always do so at the expense of the smaller
    molecule.
     Nuclei – Blue/ Black
    Cytoplasm, muscle , RBC → Red
    Collagen → Blue/green
    Applications
    • Demonstrate collagen and muscle in normal
    tissue
    • Differentiate collagen and Muscle in tumors
    • Identify an increase in collagenous tissue
    • Indicate fibrotic change in cirrhosis of liver
    • Indicate fibrotic change in pyelonephritis
    • Distinguish tumors that have arisen from
    muscle cells and fibroblasts
    Van Gieson Technique 
    • Collagen – bright red
    • Nuclei – Blue/Black
    • Cytoplasm, muscle, RBC , elastin , reticulin -yellow
    Demonstration of Reticular Fibres
    • Reticulin fibres are demonstrated either by using
    dyes as means of coloring agent or by metal
    impregnation methods

    • Silver impregnation is the best method because it


    provides good contrast enabling even the finest
    fibers to be resolved
    Reticulin Stain

    Reticular fiber – Black


    Nuclei- Gray
    Other elements- According to counterstain used
    Pigment and Minerals
    Hemosiderin- 3  methods

    1.Perl’s prussian blue reaction – for ferric ion


    2. Lillie’s method – for ferrous iron
    3. Hukill and putt’s method – for both ferric and
    ferrous iron
    Perl’s Stain
    Ferric iron –Blue
    Nuclei – Red
    Von Kossa Method for Calcium
    • Tissue sections are treated with silver nitrate solution
    • Calcium is reduced by the strong light and replaced
    with silver deposits, visualized as metallic silver

    • With the H&E stain, calcium appear deep blue-purple


    Bile Pigment: Modified Fouchet’s technique
    Bile pigment : Emerald to blue green
    Muscle :yellow
    Collagen :red
    Melanin: Masson-Fontana Stain
    Melanin – black
    Nuclei – red
    IMMUNOHISTOCHEMISTRY
    • Immunohistochemistry (IHC) combines histological,
    immunological and biochemical techniques for the
    identification of specific tissue components by means
    of a specific antigen/antibody reaction tagged with a
    visible label.

    • IHC makes it possible to visualize the distribution and


    localization of specific cellular components within a
    cell or tissue.
    IHC is an application of antibodies to tissue
    preparation for the localization of target
    antigens:

     Wide range of specific antibodies


     Highly sensitive detection system
    • Immunohistochemistry utilizes labeled
    antibodies to localize specific cell and tissue
    antigens, and is among the most sensitive and
    specific histochemical techniques

    • Because many targeted antigens are proteins


    whose structure might be altered by fixation
    and clearing, so frozen sections are commonly
    used
    PRINCIPLE
    • The principle of immunohistochemistry is to
    localize antigens in tissue sections by the use
    of labeled antibodies as specific reagents
    through antigen-antibody interactions that are
    visualized by a marker such as fluorescent dye,
    enzyme, radioactive element or colloidal gold.
    PROCEDURE
    Fixation

    1.TISSUE PREPARATION Dehydration & Embedding

    Tissue sectioning

    2.DEPARAFFINATION Dewaxing

    Rehydration
    3.ANTIGEN RETRIEVAL

    4. BLOCKING Peroxide Block


    NONSPECIFIC STAINING
    Power Block

    Antibody incubation

    5.IMMUNOSTAINING Detection system

    Colour development
    6.COUNTER STAIN

    7.CLEARING & MOUNTING

    8.VIEWING
    THANK YOU

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