LESSON 7 FIXATION

If you want a permanent sample, FIXATION !

Why Fixation? To avoid self-destruction (or autolysis) by intracellular enzymes To prevent bacterial decomposition To preserve the structure and molecular composition

the first step and the foundation in a sequence of histology

Fixation The technique in the preparation of specimens for the purpose of maintaining the existing form and structure of all the constituent elements. Chemical fixation Physical fixation

Fixation The technique in the preparation of specimens for the purpose of maintaining the existing form and structure of all the constituent elements. Chemical fixation makes cells permeable to staining reagents and crosslinks their macromolecules so that they are stabilized and locked in position using fixatives . Physical fixation

The ideal/best fixation …… Preservation in a condition identical to that existing during life. a life-like state as possible! Promptly (ASAP) and adequately

Intravascular Perfusion Facilitate the penetration of the fixative

Fixation Safe guard against the damaging To facilitate differential staining with dyes and other reagents.

Fixatives Organic solvents Heavy metal fixatives Alcohol Aldehyde precipitate proteins and other macromolecules. Aldehyde chemically cross-links the primary amines of neighboring proteins Heavy metal fixatives

The most common fixatives for routine light microscopy ? “Formalin” A buffered isotonic solution of 37 - 40% formaldehyde (by weight)

Formaldehyde HCHO causes proteins to cross link in a meshwork Lipids, nucleic acids and carbohydrates are trapped in a matrix of insoluble and cross-linked proteins milder fixative

Formaldehyde's mechanism of action (within 24 hr) (several weeks) In practical terms, formaldehyde penetrates tissues rapidly (because of its small size), but it slowly cross-links the proteins.

Methylene bridges between the side amino groups

Formaldehyde HCHO causes proteins to cross link in a meshwork milder fixative Formalin Paraformaldehyde Higher polymers (n up to 100) are insoluble in water and sold as a white powder

Formation of formaldehyde polymers Depolymerization of paraformaldehyde To be useful as a fixative, the solution must contain monomeric formaldehyde.

+10-14% MeOH as stabilizer, inhibit polymerization 1 in 10 dilution

2. Glutaraldehyde HCO-[CH2]3-CHO enormous potential for cross-linking proteins standard fixative for EM 'EM grade' glutaraldehyde

2. Glutaraldehyde HCO-[CH2]3-CHO Polymerization reaction of glutaraldehyde In solution glutaraldehyde exists as polymers of various sizes which exhibit an enormous potential for cross-linking proteins

Reaction of polyglutaraldehyde with amino groups of proteins. In contrast with formaldehyde, the chemical reaction of glutaraldehyde with protein is fast, but the penetration of tissue is extremly slower, especially for the larger oligomers. (fast)

formaldehyde vs. glutaraldehyde Fixation (cross-linking) Penetration

formaldehyde and glutaraldehyde The combination takes advantage of the rapid penetration of formaldehyde, which quickly stabilizes the structure of the tissue, followed by a more thorough cross-linking of proteins mediated by the more slowly penetrating glutaraldehyde 2-4 % paraformaldehyde + 1-2 % glutaraldehyde

3. Osmium Tetroxide (OsO4) fixative & stain binds to and stabilizes lipid as well as proteins Secondary fixative OsO4 → OsO4∙nH2O (osmium black)

Double fixation Standard procedure for Electron Microscopy 1st 2nd Special Preparation for fine structural studies

4. Ethanol C2H5OH Coagulates protein (but not nucleoprotein) Precipitates glycogen Dissolves many lipids Powerful dehydrating agent Shrinkage & hardening

5. Acetic acid pure water-free CH3COOH ‘glacial acetic acid’ Used in many fixing mixtures Precipitate nucleoprotein

6. cold Methanol or Acetone Rapid fixation (than aldehydes) Precipitating proteins and carbohydrates Fixation and dehydration occur simultaneously Possible shrinkage of the samples

Factors involved in fixation Temperature . Size of specimens and penetration of fixatives. Volume of fixatives (10 times that of the tissue). pH and buffers . Osmolality . Concentration of fixatives . Duration of fixation slow (by cold) / 37℃ (accelerated) / heat (almost inhibit, but …..)

Learning Resources Junqueira’s Basic Histology ; pp 1-3 Looking at the Structure of Cells in the Microscope http://www.ncbi.nlm.nih.gov/books/NBK26880/#A1733 “Tissues Are Usually Fixed and Sectioned for Microscopy.”

The Theory & Practice of Staining Lesson 8 Staining The Theory & Practice of Staining

Why staining ? Most cells and tissues are transparent and colorless. One way to make them visible is to stain them with dyes. Successful staining ? Specificity - selectivity, the ability to discriminate between individual tissue component Sensitivity – a capacity of the stain to demonstrate a tissue substance in low concentration

Types of staining Vital staining Elective solubility Staining with dyes Metal impregnation Production of colored substances

Types of staining Vital staining Elective solubility In living tissues/cells trypan blue (cytoplasmic phagocytosis) Janus green (mitochondria) Elective solubility Staining with dyes Metal impregnation Production of colored substances

Types of staining Vital staining Staining by Elective Solubility Lipid-rich structures (e.g. fat droplets) Lipid-soluble dyes (lipophilic dye) Frozen sections Staining with dyes Metal impregnation Production of colored substances

Sudan black stained the lipid droplets & adipose cells

Types of staining Vital staining Elective solubility Staining with dyes Basic & Acidic dyes Metachromatic Staining Local formation of a dye Metal impregnation Production of colored substances

Basic & Acidic dyes Basic /cationic dye Acidic/anionic dye Basophilic (e.g. nucleic acids, glycosaminoglycans, acid glycoprotein & other acidic structures) affinity for negatively charged molecules Toluidine blue, alcian blue, methylene blue, Hematoxylin Acidic/anionic dye Acidophilic (e.g. proteins with many ionized amino groups, mitochondria, secretory granules, collagen) Orange G, eosin, acid fuchsin No relevance to pH Positive charge (cationic) / Negative charge (anionic)

H&E (Hematoxylin and Eosin) Hematoxylin (blue-purple) DNA of the cell nucleus, RNA-rich portion Eosin (pink) Other cytoplasm, collagen Good Counterstain a stain with color contrasting to the principal stain, making the stained structure more easily visible

PAS with H&E staining

General procedure Deparaffin (Re)Hydration Hematoxylin Rinse in water Eosin Dehydration Xylene Mounting a protective glass coverslip on the slide with adhesive mounting media (Canada Balsam, Gelvatol……)

Metachromatic Staining Metachromasia To produce a color other than that of the dye used Metachromatic tissue Cartilage, connective tissue, amyloid …… Metachromatic dye Toluidine Blue orthochromatic color nucleic acids blue metachromatic color sulfated polysaccharides purple

Local formation of a dye Feulgen stain DNA Periodic acid-Schiff(PAS) stain carbohydrate   

Feulgen stain DNA can be specifically identified and quantified Mild hydrolysis with HCl frees the aldehyde group of deoxyribose in DNA, which is then reacted with the Schiff's reagent.

Periodic acid-Schiff(PAS) stain for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) Periodic acid Schiff reagent an aqueous solution of rosaniline and sulfurous acid iodic(VII) acid, HIO4 or H5IO6

Periodic acid-Schiff(PAS) stain periodic acid is to oxidize some of the tissue carbohydrates. This produces aldehyde groups, which can then condense with Schiff's reagent forming a bright purple or magenta coloration. +  HIO4  =   + 2(R-CHO) =

Types of staining Vital staining Elective solubility Staining with dyes Metal impregnation e.g. silver staining A common method of visualizing certain ECM fibers & specific cellular elements in nervous tissue Production of colored substances

Learning Resources Junqueira’s Basic Histology ; pp 3-4 Looking at the Structure of Cells in the Microscope http://www.ncbi.nlm.nih.gov/books/NBK26880/#A1735 “Different Components of the Cell Can Be Selectively Stained ”

LESSON 9. HISTOCHEMISTRY & CYTOCHEMISTRY كيمياء خلايا وأنسجة الجسم البشري الكيمياء الخلوية

Lecture Overview What’s histochemistry / cytochemistry ? Principle (Enzyme Reaction) Procedure Examples

Histochemistry & Cytochemistry Staining Enzyme histochemistry Immunohistochemistry

Enzyme histochemistry Methods for localizing cellular structures using unique enzymatic activity present in those cell structures. e.g. Lysosome : acid phosphatase Membrane : alkaline phosphatase Mitochondria : dehydrogenase

Enzyme Reaction Substrate: the substance acted upon by an enzyme Speed up Substrate: the substance acted upon by an enzyme

Preparation of tissues for enzyme techniques To preserve enzymatic activity Fresh unfixed or mildly fixed tissue Rapid disappearance of most enzymes after death Cryostat Speed, ease of handling, and versatility

Procedure Incubate in Substrate solution Enzymatic action on the substrate Formation of primary reaction product (PRP) Contact with a Marker compound (Trapping agent) Reacts with a PRP contains a visible reaction product such as a heavy metal or a colored reaction product. Final reaction product (FRP) Insoluble Visible by microscopy Color, electron-dense material, precipitates Trapping agent - binds to the substrate and contains a visible reaction product such as a heavy metal or a colored reaction product.

Types of Methods Simultaneous capture Post-incubation coupling Production of the PRP and its capture as a coloured FRP take place simultaneously in one incubating medium Post-incubation coupling Two reactions are carried out separately one after another Production of a colourless PRP first Subsequently coupled into a coloured FRP in a separate solution.

Phosphatases Alkaline phosphatase Acid phosphatase : removes a phosphate group from its substrate Alkaline phosphatase Acid phosphatase pH 9.0 Kidney, intestine, …… ≈pH 5.0

Dehydrogenases mitochondria Remove hydrogen from a substrate and transfer it to a hydrogen receptor mitochondria

Peroxidase myeloperoxidase Promotes the oxidation of certain substrates with the transfer of hydrogen ions to hydrogen peroxide, forming molecules of water myeloperoxidase Diagnosis of certain leukemia (cancer of blood)