Histochemistry OBJECTIVES: . Understand the principles behind microscopy . Understand the different staining techniques and their uses MICROSCOPY . Light microscope . Electron microscope . Scanning EM . Transmission EMOptical resolution — capacity of an optical system to reveal detail in a specimen 1) Scanning electron microscopy (SEM) and back-scatter scanning electron microscopy (BSEM): * Evaluate bone structures + Examining the vascular structure of various corrosion-casted tissues, including bone, muscle, joint, ligament, and tendon. The limitations of SEM ar: * The specimen must be dried before observation causing distortion of the original spatial structure and morphology (The problem seems to have been solved by the new low-temperature or cryo-SEM system) * Limited specimen size2) Transmission Electron Microscopy: (TEM) ° New * High-voltage electron microscopic tomography method * Also has direct 3D imaging capability. Used to view the structural relationships between collagen and mineral in bone. * Most powerful method for evaluating the ultrastructure and morphology of large molecules (such as proteoglycans or collagens), subcellular components, cells, and even the implant-tissue interface.Histochemistry Procedures « “Soft tissue sample * Fixation * Dehydration * Embedding * Sectioning * Staining The structure of a tissue is determined by the shapes and sizes of macromolecules in and around cells. The principal macromolecules inside a cell are proteins and nucleic acids.Fixation +An essential part of all histological and cytological techniques is preservation of cells and tissues as they naturally occur. To accomplish this, tissue blocks, sections or smears are usually immersed in a fixative fluid. © Fixation is commonly achieved by immersing the tissue in a suitable fixative solution * fixative solution penetrates the tissue and biologically inactivates proteins by creating conformational changes in the tertiary structure. * a physiochemical process in which cells or tissues are fixed chemically. * Asa result, the tissue or cell can combat the successive treatment by different reagents with negligible disfigurement of morphology.eAn ideal fixation involves complicated progression of chemical episodes. An ideal fixative is presumed to transmit mechanical toughness to tissue so that it resists destruction due to further processing steps. It prevents the autolysis, putrefaction of tissue as well as tissue component degradation. Fixation should be able to preserve the cellular structure and tissue architecture in life-like manner Fixation amends the physio-chemical state of tissues so that it remodels the reactiveness of cellular components for stainsFixatives can be classified in different ways, as shown: Table 1 Classification of fixatives based on chemical composition Fixatives Examples 1. | Physical agents Heat. microwaves 2. | Aldehydes Formaldehyde, acrolein, glutaraldehyde 3. | Coagulants ‘ohol. ethyl alcohol, id 4. | Oxidizing agents m tetroxide: 5. | Miscellaneous Picric acid, mercuric chloride Table 2 Classification of fixatives based on number of struc- 1. | Simple fixatives e.9.. For 2. | Compound fixatives | ¢.g.. Bouin's fluid, formol saline, Zenker’s fluidTable 3 Classification of fixatives based on type of structures fixed Fixatives Examples 1, | Histochemical | Formaldehyde, glutaraldehyde, vapor fixatives fixatives 2. | Microanatom- | Bouin's fluid, 10% formalin, Zenker's ical fixatives | fluid, formol calcium, Heidenhain's susa, Helly's fluid, Rossman’s fluid, 3. | Cytologic Champy’s fluid, glacial acetic acid, fixatives alcohol, formol saline, Carnoy’s fluid, Clarke's fluid, Newcomer's fluid, Flemming's fluidFunctions of Fixative: If a fresh tissue in kept as such at room, temperature it will become liquefied with a foul odour mainly due to action of bacteria i.e. putrefaction and autolysis Most aim of fixation is 1. To preserve the tissue in as It like manner as possible. 2. To prevent postmortem changes like autolysis and putrefaction.Prevent autolysis (enzymes attack) [by inactivating lysosomal enzymes] o Autolysis seems to be a frequent issue in enzyme-rich tissues, o (rigorously autolyzed tissue does not get stained properly). Autolysis is the lysis or dissolution of cells by enzymatic action probably as a result of rupture of lysosomesPrevent putrefaction (bacterial attack) of tissues [inhibit the growth of bacteria and molds]: o bacterial invasion also can be blocked by following the strict antiseptic methods. Putrefaction The breakdown of tissue by bacterial action often with formation of gas.a Conserving the association in between cells and extracellular substances. * Stabilize the cell component by making them insoluble, * Preventing osmotic damage of tissue, which may cause shrinkage or swelling, thus preserving the cellular and tissue structure in life-like state. * Making tissue firm [Hardening] (so that gross cutting becomes much easier). * Fixatives help make the tissue more easily permeable for subsequent reagents * help in increased visibility of different elements of tissue. * The hardening effect of fixatives allows easy manipulation of soft tissue like brain, intestines etc.* Optical differentiation: it alters to varying degrees the refractive indices of the various components of cells and tissues so that unstained components are more easily visualized than when unfixed. * Fixation, especially in organic liquids that dissolve or disrupt the lipids of the cell membrane, allows relatively large molecules to penetrate and escape. Furthermore, the cytoplasm becomes permeable to macromolecules, forming a proteinaceous network sufficiently porous to allow further penetration of large molecules. In this context, “large molecules” include those of paraffin wax, those of antibodies used for immunostaining, and larger dye molecules.Factors Affecting Fixation and Fixatives: Fixation profoundly affects histological and immunohistochemical staining, technicians, pathologists and research workers must therefore decide on the most appropriate method. Aspects to consider are temperature, size of the storage container, volume ratio, salt concentration, pH and incubation time.Length of Fixation: The ideal time of fixation is experimentally determined for different types of tissue. If time period for fixation is longer, > © 1% over—cross-linking, * Samples become brittle. If time period for fixation is short, > * Sufficient amount of penetration in tissues and cross-linking will not occur. * For oral soft tissue, overnight fixation is sufficient.Temperature: * Temperature of fixative during fixation may affect the tissue architecture. * Rate of fixation is increased with increase in temperature, * But: o increased temperature ~ increase autolysis rate. o low or decreased temperature > J) diffusion rate > results in extended penetration time. * For electron microscopic studies, 0° to 4°C is appraise as ideal temperature.Concentration ¢ If fixative agent concentration is low > need prolonged time for fixation. ¢ If fixing agent concentration is high ~ it results in damaging of cellular structures as well as obliterated enzyme activities. Different fixatives have different ideal concentration that is determined experimentally; for example, ideal fixative for oral soft tissue is formalin used in 10% concentrated solution.Size Tissue thickness is one of the important factors for fixation. * Large sample size > Unfavourable for the fixative to penetrate and reach to the deeper part of the tissue > which would result in autolysis of epithelium. * Ideally 4- to 6-mm-thick specimen is best for complete penetration by fixatives. Note : If the specimen is large then see that the sections are made to make slices which have a thickness of less than 1.5 cm so that fixative can penetrate the tissue easily.Osmolarity ¢ It is important that the concentration of fixative is isotonic or hypotonic ¢ If osmolarity of tissue as well as fixative is same, it will prevent swelling or shrinkage of the tissue.Properties of fixatives 1. Penetration Fi nis done by immersing the tissue in fluid containing the fixative. Faster a fixative can penetrate the tissue better it is penetration power depends upon the molecular weight e.g. formalin fixes faster than osmic acid. 2. Solubility of fixatives - All fixatives should be soluble in a suitable solvent, preferably in water so that adequate concentrations can be prepared. 3. Reaction - Most fixatives are acidic. It may help in fixation but can affect staining so has to be neutralized e.g. formalin is neutralized by adding of calcium carbonate.Amount of fixative The fixative should be at least 15-20 times the bulk of tissue. For museum specimens the volume of fixative is > 50 times.Various Fixating Agents Used in Histopathology: Formaldehyde or Formalin: The most routinely used solution for fixation, 10% formalin solution v/v Aqueous suspension of formaldehyde (which is a gas) In 10% neutral buffered form, formaldehyde is found to be the most commonly used fixative in pathology. Formalin also contains about 10% methanol to retard the formation of higher polymers, which eventually fall out of solution as paraformaldehyde. Polymers of up to 100 repeat units are termed “paraformaldeyhde” and are insoluble.After continuous storage for long periods, accumulations of white deposits (of paraformaldehyde) are observed in the solution. [Also, storing formalin at low temperature UW white deposits] However, in order to penetrate tissues and work effectively as a fixative, working formaldehyde solutions need to consist predominantly of monomeric methylene hydrate. This involves the monomerization of the polymerized form. This is commonly achieved by diluting formalin to 10% (v/v) by using a buffer of physiological pH. Reaction between the formaldehyde and macromolecules of tissue seems to be complex: + Formaldehyde reacts with nucleic acids as well as proteins + it penetrates between nucleic acids and proteins and forms stabilized shell of nucleic acid-protein complexFormaldehyde causes: (As compared with other fixatives) * lesser tissue shrinkage, with exceptions being acetone and ethanol. © more hardening of tissue * Conservation of lipids, but carbohydrates are not fixed by formaldehyde.Glutaraldehyd * Glutaraldehyde was found in 1963 by Sabatini et al as particular fixative for ultrastructural researches. Glutaraldehyde = 2 aldehyde groups that are divided by 3 methylene bridges. + Penetration rate ~> slower when compared with formaldehyde. + When polymerization of aqueous solution of glutaraldehyde occurs, it forms oligomeric and cyclic compounds, + Forms glutaric acid by oxidation. For stability, it requires pH of 5 and storage at 4°C. + At room temperature, glutaraldehydes are not able to cross-link the nucleic acids Glutaraldehyde preserves the ultrastructure of the tissue, thereby it is used in electron microscopy studies, but owing to poor penetration and overhardening properties, it is not used as tissue fixatives for light microscopy. + On exposure to oxygen, glutaraldehyde becomes unstable and breaks down with decrease in pH. Glutaraldehyde can act as sensitizer, and its exposure may result in respiratory tract, skin, and digestive tract irritation.Osmium Tetroxide: + water soluble fixative * also soluble in nonpolar solvents + React with proteins side chains that cause cross-linking. + The reactive groups of osmium tetroxide include various groups such as disulfide, carboxyl, hydroxyl, sulfydryl, amide, and so on. + Due to slow rate of reaction + restricted penetration into tissue > large amounts of carbohydrates as well as proteins are eradicated. + For electron microscopic studies, osmium tetroxide is used as secondary fixative, and it also performs well as stain and imparts contrast when observed under electron microscope. + Helpful for staining of lipids in frozen sections. + Causes swelling in tissue, which can be decreased by adding sodium chloride or calcium chloride to fixatives. * Sold as: crystalline solid that is sealed in glass ampule. ‘+ Crystals convert from solid state to vapor state. + Continued exposure to osmium tetroxide vapors can cause deposition into cornea, which eventually leads to blindness.Mercuric Chloride: * Used as a tissue fixative for histopathology. * It chiefly reacts with cysteine and also reacts with amines, amides, sulfydryl groups, and ammonium salts, and results in tissue hardness. + Itacts as a strong protein coagulant. + With acid dyes, it shows strong staining affinity. It also reacts with phosphate remnants of nucleic acids and adequately fixes the nucleoproteins. Therefore, because of these reasons, it is observed that mercuric fixatives are the major component of some fixatives such as Helly’s fixative and b-5 fixatives. + Nowadays, mercurial fixatives are not routinely used except for fixation of hematopoietic tissues. © They are toxic in nature © should not be allowed to come in contact with metals. © have slow penetration capacity, so the thickness of the specimens being fixed by mercuric fixatives should be thin.Glyoxal + Also known as ethanedial or oxalaldehyde. * Glyoxal is considered as alternative fixative to formalin because it is a dialdehyde in nature. * It is a bifunctional aldehyde. * potentially reactive aldehyde -> also causes cross-links. * Glyoxal fixed tissues may demonstrate precise cellular details, lysed erythrocytes, and disintegrated microcalcifications. * Does not evaporate from the solution at room temperature > less dangerous in use than formaldehyde. * For microwave fixation, glyoxal is the chief component used in the fixatives. * By molecular weight, glyoxal is the third smallest aldehyde after formaldehyde and acetaldehyde. It contains two carbon atoms. * Commercially manufactured as aqueous solution that contains hydrates such as trimers, dimmers, and ring structures.Picric Acid © Coagulant fixative. © It forms picrates with basic protein groups, which causes coagulation. * Not used for demonstration of DNA or RNA * Can hydrolyze nucleic acids. * Disintegrate calcium deposits in samples. « Although picric acid is not able to fix most carbohydrates and lipids * Most advised fixative to preserve glycogen. Brighter staining is seen by picric acid fixatives. * Acidic solution * Gets washed out by alcohol. To avoid this, lithium carbonate is added, which acts as a neutralizer. * Luna reported that if picric acid is present in the tissue or not completely removed, distortion or obliteration of cellular structures will occur as outcome.Ethanol and Methanol «For ethanol and methanol, fixation initiates at 50 to 60% concentration and greater than 80% concentration, respectively. * Coagulants: cause protein denaturation. © They cause interruption in hydrogen and hydrophobic bonding by substituting water in tissue environment, which results in change in tertiary structure. * Ethanol causes mis-presentation of cytoplasmic as well as nuclear details, * But sometimes: Used for preservation of glycogen. * More commonly used for fixation of exfoliative cytology smears and blood films.Acetone ¢ efficacious lipid solvent that results in tissue brittleness. ¢ Apart from tissue fixation, they are primarily used as an agent for dehydration in tissue processing. * not recommended for use in automatic tissue processor, Because: o extremely volatile o flammableAcetic Acid * Non-coagulative fixative ¢ Causing nuclear proteins coagulation. Stabilizes and assists to prevent nucleic acids loss. ¢ in combined with ethanol, is used as an effective cytological fixative © Results in swelling of cells. ¢ Time required for fixation by acetic acid is less as penetration of acetic acid is faster into tissuesPotassium Dichromate ¢ Non-coagulant fixative ¢ But if used in combination with acid solution, it acts as a coagulant fixative. ¢ Seldom used alone for fixation because chromate ions will link with few lipids and makes them insoluble. * It conserves mitochondria but dissolves DNA. ¢ Tissues that are fixed with chromate fixatives have to be washed completely in water before processing of tissues any further. This step is important as it avoids establishment of chromate suboxide that is insoluble.Bouin’s Fixative + Non-coagulant picrate fixative solution * good fixative for conserving delicate soft tissue structures. ¢ Contains: picric acid with little quantity of acetic acid as well as formaldehyde. © Cannot be used because it decreases the severity of hybridization.Acrolein -is a three carbon aB unsaturated monoaldehyde. ¢ Acrolein provides magnificent preservation of structural detail and conserves the virus antigenicity. ¢ known as acrylic aldehyde. Reversibly cross-links macromolecules. ¢ Not commonly used because: it is unstable at alkaline pH and forms insoluble polymers. ¢ Highly reactive and penetrate tissues rapidly. * Acrolein fixatives are chiefly used in enzyme histochemistry.Genipin + Glycone derivative * Cross-linking agent * cross-linking with hydroxylysine and lysine. © reacts with amino acids form dark blue color pigment. Fixed tissue becomes resistance against collagenase degradation ¢ Available as: a crystalline white powder that is soluble in acetone, methanol, and ethanol. * works at pH 7.4-8.5. © Biomedical applications: such as dentistry, articular cartilage tissue engineering applications, nerve regeneration, ...