Microtechnique 1

Prof. Dr. Magda Ismail Youssif Histochemistry and Cell Biology Department Medical Research Institute Alexandria University HISTOTECHNIQUES

The Techniques for processing the tissue ,whether biopsies, surgical excision ,or tissue from autopsy postmortem .So as to enable the pathologist or cytologist to study them under the microscope

Histology is the study of the microscopic anatomy of cells and tissues of plants and animals . In the study of histology ,there are two important considerations with regard to methodology 1.The type of microscope. 2.The preparation of the tissue or organ from human or animal in a manner suitable for viewing under the microscope The technique of getting tissue into paraffin is called tissue processing. Animal handling

Animal research has been directly responsible for most advances in medicine (human and animal) and will continue to be important for many years to come KILLING AND FIXATION Before tissues can be dissected out for examination the animals must be immobilized (to be stable). Usually an anesthetic such as chloroform, ether, or Nembutal is satisfactory. If delicate cell organelles (mitochondria) are to be studied, the animal should be killed by a blow on the head. The organs to be studied should be removed immediately from the body and placed in an appropriate fixative solution. The sacrificed animal should be laid out ventral side up on the board and pinned out through the fore and hind feet. The first step in the dissection is to lay back the skin. This is done by pinching a skin fold in the midventral line and making a cut, which is continued forwards to the level of the lower lip and backwards around the genitals. The lower scissor blade (which is within the skin) must be kept horizontal so as not to damage the body wall beneath. The skin should be pinned back...... which should be extended Entry to the abdominal down to the genitals and cavity is gained by forward as far as the xiphoid pinching the wall and cartilage. making a small cut..... The abdominal wall should be cut along the line of the thorax, which is defined by the lower ribs.

11 Make up the cell membrane

12 13 14 15 16 Fresh tissue can be examined by: Processing of tissue : Isolate cells for culture Process into paraffin blocks Freeze for protein, lipid, sugar, Process for EM DNA/RNA etc.extracts

Freeze for histology/histochemistry/ -Fix & use for immunohistochemistry -Dehydrate -Infiltrate with xylene -Infiltrate with hot paraffin wax -Make blocks for sections -Store at room temperature Dry ice in 2-methyl butane

OCT in plastic mold Frozen or paraffin tissue can then be sectioned for histology 3--30 micron sections Tissue Processing

Tissue specimens received in the surgical pathology laboratory have a request form, that lists the patient information and history along with a description of the site of origin. The specimens are accessioned by giving them a number that will identify each specimen for each patient .

The technique of getting tissue into paraffin is called tissue processing. The main steps in this process are fixation dehydration clearing and embedding Steps vary according to : -types of tissue & microscopy structure to be seen - stains to be used -time duration etc. paraffin section preparation Specimen: as fresh as possible Labeling & Numbering Fixation: to preserve the structural organization Dehydration: replace the water in the tissue by alcohol Clearing: replace the alcohol by xylene Embedding: replace the xylene w/ paraffin Sectioning & mounting

Fixation

•Fixation is defined as the rapid preservation of tissue components in order to arrest cellular processes and to maintain, as close as possible, a resemblance to the living condition.

•Thus the purpose of fixation is to preserve tissues permanently in as life-like a state as possible. • Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis.

• A variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated. Fixatives functions Fixatives perform a number of important functions.. 1- They prevent post-mortem autolytic, osmotic, bacterial, and other changes in the tissue. 2-They render the substances of the cell insoluble. 3- They bring out differences in the refractive indices of cellular components and thus make them more readily visible. 4. - fixation also resists tissue degradation by endogenous (enzymatic autolysis) or exogenous (bacterial action) mechanisms 5- Fixatives coagulate tissue proteins as to prevent loss of diffusible substances 6- fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis. 7- a fixative typically protects a sample from extrinsic damage. 8- Stabilizes tissue proteins to prevent further changes, such as decay 9- To leave tissue in a condition which facilitates differential staining with dyes and other reagents.

The choice of a particular fixative will depend on the tissue and the specific techniques which are to follow fixation Ideal Fixative must be : 1. Do not shrink, swell or harden tissue substrates 2. Do not dissolve tissue components 3. Kill microorganisms 4. Retain the original form of tissue constituents throughout subsequent processing of tissue 5. Compatible with subsequent staining method 6. Adequate penetration rate 7. Should be non toxic and non allergic 8. Preserve tissue in their natural state and fix all chemical cell components (protein, carbohydrates, fats etc.,) 8, Not react with the receptor sites . Types of fixation process

There are three types of fixation process: 1- Heat fixation: After a smear has dried at room temperature, the slide is passed through the flame of a Bunsen burner several times to heat ,kill and adhere the organism to the slide. Heat-fixation method can be successfully used for preparing Gram- negative and Gram-positive bacteria samples for studies 2- Perfusion: Fixation via blood flow. The fixative is injected into the heart, then spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms) 3- Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 15-20 times greater than the volume of the tissue to be fixed (this is a chemical fixation). The fixative must diffuse through the tissue to fix, so tissue size and density, as well as type of fixative must be considered. Using a larger sample means it takes longer time for the fixative to reach the deeper tissue. This requires a chemical fixative that can stabilise the proteins, nucleic acids and mucosubstances of the tissue by making them insoluble. Fixation mechanisms

Crosslinking fixatives •Aldehydes •Oxidising agents Precipitating fixatives •Ethanol •Methanol •Acetone Other fixatives •Mercurials(B-5) •Zenkers fixative •Picrates •(HOPE) Fixative Cross linking fixatives Cross linking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and adds additional rigidity to the tissue. These tend to preserve the secondary structure of proteins Ccosslinking fixatives includes : 1- Aldehydes Popular Aldehydes for Fixation : Formaldehyde Glutaraldehyde

2- Oxidising agents The oxidising fixatives can react with various side chains of proteins and other biomolecules, allowing the formation of crosslinks which stabilise tissue structure Aldehydes (Crosslinking fixatives) • Aldehydes include formaldehyde (formalin) and glutaraldehyde. • Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins, so that antigenicity is not lost. • Therefore, formaldehyde is good for immunoperoxidase techniques. • Formalin penetrates tissue well, but is slow. • The standard solution is 10% neutral buffered formalin. • Glutaraldehyde • causes deformation of alpha-helix structure in proteins so is not good for immunoperoxidase staining. • However, it fixes very quickly so is good for electron microscopy. • It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. • The standard solution is a 2% buffered glutaraldehyde Oxidizing agents - The oxidizing fixatives can react with various side chains of proteins and other biomolecules, allowing formation of cross links that stabilize tissue structure. - Disadvantage: they cause extensive denaturation - Advantage: preserving fine cell structure and are used mainly as secondary fixatives. Oxidizing agents include • Osmium tetroxide (is often used for electronic microscopy) • Permanganate fixatives (potassium permanganate). • Dichromate fixatives (potassium dichromate). Precipitating fixatives (Denaturing fixatives) Precipitating (or denaturing yb tca sevitaxif ) dna selucelom nietorp fo ytilibulos eht gnicuder eht gnitpursid yb )netfo(hydrophobic rieht snietorp ynam evig hcihw snoitcaretni .erutcurts yraitret The precipitation snietorp fo noitagergga dna gniknilssorc eht morf ssecorp tnereffid yrev a si .sevitaxif edyhedla eht htiw srucco hcihw The most common precipitating fixatives are ethanol , methanol.and Acetone is also used. Alcohols (Precipitating fixatives) • Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol). • They are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. • However, they are very good for cytological smears because they act quickly and give good nuclear detail. Acetic acid is a denaturant that is sometimes used in combination with the other precipitating fixatives . The alcohols era ,sevlesmeht yb , eussit fo egaknirhs esuac ot nwonk noitaxif gnirud while acetic acid detaicossa si enola ;gnillews eussit htiw combining ni tluser yam owt eht eussit fo noitavreserp retteb morphology Other fixatives

•Mercurials(B-5)

•Zenkers fixative

•Picrates

•(HOPE) Fixative Mercurials • They contain mercuric chloride and include such well-known fixatives as B-5 and Zenker's. • These fixatives penetrate poorly and cause some tissue hardness, • but are fast and give excellent nuclear detail. • Their best application is for fixation of hematopoietic and reticuloendothelial tissues. • Since they contain mercury, they must be disposed of carefully . B-5 is: Water 90 ml Formalin (40% HCHO) 10 ml Mercuric chloride 6 g Sodium acetate (anhydrous) 1.25 g The sodium acetate brings the pH into the 5.8-6.0 range. Fix by immersion, 12-24 hours, then transfer to 70-80% alcohol Picrates • Picrates include fixatives with picric acid. Foremost among these is Bouin's solution. • It does almost as well as mercurials with nuclear detail but does not cause as much hardness. • Picric acid is an explosion hazard in dry form. As a solution, it stains everything it touches yellow, including skin HOPE Fixative Hopes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) gives formalin-like morphology. excellent preservation of protein antigens for immunohistochemistry and enzyme histochemistry. good RNA and DNA yields and absence of crosslinking proteins. Classification of Fixatives & Fixing fluids

Physical Chemical

 Heat Fixation  Microwave simple compound fixation  Freeze drying fixation Microanatomical cytological Histochemical

Nuclear Cytoplasmic

Simple fixatives or primary fixatives or unmixed fixatives : Formalin Ethyl alcohol • Colourless liquid • non-coagulant fixative • Reducing agent • Acidic, cheap Osmium tetroxide • easy to prepare • Strong oxidizing agent • Expensive, poor penetration • relatively stable Potassium dichromate Mercuric chloride • Strong fixative • Coagulant fixative • Fix lipids Trichloro acetic acid • black precipitate in tissues • Protein precipitant Glacial acetic acid Picric acid • Protein precipitant • Protein precipitant • Colourless solution • Used as saturated solutions • Pungent smell Compound fixatives or fixation mixtures : Compound fixatives

Micro- Cytological anatomical fixatives fixatives

Nuclear fixatives

Cytoplasmic fixatives Micro-anatomical fixatives : Used to preserve the anatomy of tissue

10% formal saline

10% neutral buffered formalin

Heidenhain’s Susa Formal-sublimate Formal-saline sublimate Zenker’s fluid Helly’s fluid Bouin’s fluid Gendre’s fluid Cytological fixatives: used to fix intracellular structures 1-Nuclear fixatives :

• Chromic acid (1%) -15 ml • Aqueous osmium tetroxide (2%)- 4 Flemming’ ml s fluid • Glacial acetic acid-1ml

• Absolute alcohol - 60 ml • Chloroform – 30 ml Carnoy’s • Glacial acetic acid – 10 ml fluid 2-Cytoplasmic fixatives :

Flemming’s fluid • Chromic acid (1%) – 15 ml without acetic • Aqueous osmium tetroxide acid (2%)-4 ml

• Mercuric chloride – 5 gm • Potassium dichromate -2.5 gm Helly’s fluid • Sodium sulphate – 1.0 gm • Distilled water -100 ml • 5 ml of 40% formalin before use

Formal saline • Formal 10% • Sodium chloride histochemical fixatives used to demonstrate the chemical components of the cells Target Fixative of Choice Fixative to Avoid Osmium Tetroxide Proteins Neutral Buffered Formalin, Paraformaldehyde Chemical Enzymes Frozen Sections Fixatives Lipids Frozen Sections*, Alcoholic fixatives, Neutral Buffered Nucleic Acids Glutaraldehyde/Osmium Tetroxide Formalin Mucopolysacch Aldehyde fixatives arides Alcoholic fixatives, HOPE Chemical fixatives Biogenic Frozen Sections Amines Bouin Solution~, Neutral Osmium Tetroxide Glycogen Buffered Formalin Alcoholic based fixatives

factors affecting fixation

• There are a number of factors that will affect the fixation process: • Buffering • Penetration • Volume • Temperature • Concentration • Time interval Buffering Fixation is best carried out close to neutral pH, in the range of 6-8. Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity. Acidity favors form formalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered with phosphate at a pH of 7 . Penetration Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant. Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. One way to get around this problem is sectioning the tissues thinly (2 to 3 mm). Penetration into a thin section will occur more rapidly than for a thick section volume The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Obviously, we often get away with less than this, but may not get ideal fixation. One way to partially solve the problem is to change the fixative at intervals to avoid exhaustion of the fixative. Temperature

Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue. Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor . Concentration Concentration of fixative should be adjusted down to the lowest level possible, because you will expend less money for the fixative. Formalin is best at 10%, glutaraldehyde is generally made up at 0.25% to 4%. Too high a concentration may adversely affect the tissues and produce artifact similar to excessive heat. Time interval

Also very important is time interval from of removal of tissues to fixation. The faster you can get the tissue and fix it, the better. Artifact will be introduced by drying, so if tissue is left out, please keep it moist with saline. The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artifactual clumping will occur. Fixatives - general usage

There are common usages for fixatives in the pathology laboratory based upon • the nature of the fixatives. • the type of tissue, and • the histological details to be demonstrated. • Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced. • Neutral buffered formalin ( NBF) is a great general fixative; it is compatible with most stains. • Generally tissues are fixed from 6 to 48 hours in these solutions . • Formalin is a saturated aqueous solution of the gas, formaldehyde; this turns out to be 37-40%. It is often used simply as a 10% solution (Note: a 1:9 dilution is 10%, not the 4% you might expect.) Formalin Solution (10%, unbuffered): Formaldehyde (37-40%) ------10 ml Distilled water ------90 ml

Formalin Solution (10%, buffered neutral): Formaldehyde (37-40%) ------100 ml Distilled water ------900 ml NaH2PO4 ------4.0 g Na2HPO4 (anhydrous) ------6.5 g Mix to dissolve. Zenker's fixatives are recommended for reticuloendothelial tissues including lymph nodes, spleen, thymus, and bone marrow. Zenker's fixes nuclei very well and gives good detail. However, the mercury deposits must be removed (dezenkerized) before staining or black deposits will result in the sections. Bouin's solution is sometimes recommended for fixation of testis, GI tract, and endocrine tissue. It does not do a bad job on hematopoietic tissues either, and doesn't require dezenkerizing before staining . Bouin's are widely used fixatives for routine histological studies Tissue is fixed in either warm or cold solutions for 12-24 hours. Longer time is not harmful . They may be injected. Store tissue in 70% alcohol. No special washing out required. Removal of the yellow color in several changes of 50% alcohol is advisable before staining, but this color leaches out during procedures in preparation of sections. Bouin Solution Picric acid (saturated) ------75 ml Formaldehyde (37-40%) ------25 ml Glacial acetic acid ------5 m Mix well .For raoutine surgical material, especially for preserving soft and delicate structures such as brain tissues . Brasil's has been much used for glycogen fixation. It gives moderately fine cytoplasmic precipitation and well differentiated nuclei. Tissue is of good consistency for dissection and sectioning. Mitochondria are destroyed. These fixatives are not very suitable for most histochemical procedures (except glycogen) . 3. Carnoy's and Clarke's Fluids

Fix tissue 30 minutes to an hour in cold solution. Longer times are less satisfactory. No special washing out necessary. Store tissue in 95% alcohol. These penetrate very rapidly. Staining is excellent after both, cytoplasm is rather coarsely precipitated, nuclei well differentiated, mitochondria destroyed. Most histochemical procedures can be used after these fixatives. Carnoy Solution:

Ethanol (absolute) ------60 ml Chloroform ------30 ml Glacial acetic acid ------10 ml Mix well . Note: Used for fixation of DNA, RNA, Nissl granules and glycogen. Glutaraldehyde

is recommended for fixation of tissues for electron microscopy. The glutaraldehyde must be cold and buffered and not more than 3 months old. The tissue must be fresh and sectioned within the glutaraldehyde at a thickness of 1 mm to enhance fixation . • Alcohols, specifically ethanol, are used primarily for cytological smears. • Ethanol (95%) is fast and cheap. • Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned, there is no problem from induced brittleness.

• For fixing frozen sections, you can use just about anything--though methanol and ethanol are the best . DEHYDRATION • Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin. • First, the water from the tissues must be removed by dehydration. • This is usually done with an ascending series of alcohols, say 70% to 95% to 100%. • Sometimes the first step is a mixture of formalin and alcohol. Various dehydrating agents are satisfactory. Absolute ethyl alcohol Acetone Tertiary butyl alcohol Isopropyl alcohol Methyl alcohol, and Dioxane have been used. Dioxane and absolute alcohol are the two most widely used dehydrating agents. Some dehydrants have major disadvantages.

Acetone is very fast, but a fire hazard, so is safe only for small, hand-processed sets of tissues.

Dioxane can be used without clearing, but has toxic fumes . Dehydration Procedure

1. If tissue is in water, it should be transferred to 30% alcohol for 1-2 hours. 2. 50% alcohol for 1-2 hours 3. 70% alcohol for 1-2 hours. 4. 95% alcohol for 1-2 hours. If tissue has been stored in 70% or 90% alcohol, transfer it directly to 95% alcohol 5. 100% alcohol for 1-2 hours 6. 100% alcohol for 1-2 hours 7. The tissue should now be dehydrated and ready for "clearing" or removal of the alcohol by replacement with toluene or xylene clearing

The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). This is the process of replacing the absolute alcohol with clearing agent. • The commonest clearing agent is xylene. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive than xylene. • Chloroform can be used, but is a health hazard, and is slow. • Methyl salicylate is rarely used because it is expensive. • Benzene, cedar wood oil, tertiary butyl alcohol, or isobutyl alcohol can also used. Clearing Procedure :

1. Transfer tissue from 100% alcohol to a mixture of 1 part xylene:3 parts 100% alcohol for 1 hour. 2. Change to 1 part xylene: 1 part 100% alcohol for 1 hour 3. Change to 3 parts xylene: 1 part 100% alcohol for 1 hour 4. 100% xylene - 3 to 4 hours; tissue should clear 5. 100% xylene - 3 to 4 hours 6. After clearing, tissue is ready for infiltration Paraffin infiltration and Embedding • Finally, the tissue is infiltrated with the embedding agent, almost always paraffin. This will serve to support the tissue elements while thin sections are cut. • Paraffin is soluble in xylene, especially when it is warm, so mixtures of the two are used to slowly replace all the xylene with paraffin(melted). Waxes may be : Paraffin wax Paraplast Paraplast plus Gelatin and Celloidin Paraffins differ in melting point, a paraplast contains added plasticizers that make the paraffin blocks easier to cut. Infiltration Procedure : 1. Transfer the tissue from the clearing agent to a warm mixture of 1part paraffin: 2 parts xylene. Stopper tightly and keep in the 45° oven for 1 hour. 2. Transfer tissue to a second vial containing a warm mixture of 2 parts paraffin: 1 part xylene. Again cover and incubate at 45°° for 1 hour. 3. Transfer to the first bath of pure paraffin. Leave 2-4 hours. 4. Transfer to the second paraffin bath. Leave 2-4 hours. 5. Embed Remove the water & replace with wax-solvent Imbed the oriented specimen in molten wax

50 % ethanol 70 % ethanol 95 % ethanol 100 % label Fresh tissue ethanol Benzene/ 10% Formalin Xylene fixative Miscible with ethanol; paraffin dissolves wax wax Sectioning Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome. The microtome is nothing more than a knife with a mechanism for advancing a paraffin block standard distances across it. A very sharp knife is an important necessity for proper sectioning • For transmission electron microscopy, Plastic blocks (methacrylate, araldite, or epon) are sectioned with glass or diamond knives to about 1 micron..

Thin sections for electron microscopy (1/4 micron) are best done with a diamond knife which is very expensive MOUNTING SECTIONS ON SLIDES Once sections are cut, they are floated on a warm water bath that helps remove wrinkles. Then they are picked up on a glass microscopic slide . The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide. •If this heat might harm such things as antigens for immunostaining, then this step can be bypassed and glue-coated slides used instead to pick up the sections Use of section adhesives

An adhesive may be used, it may be smeared in a thin layer on the slide before the sections placed on slides. Several adhesives are available. The most famous is the Glycerol-Albumin Mixture For some stains an adhesive may not be advisable (e.g. Alizarin Red S calcium stain) so that sections may be simply expanded on warm distilled water on the slide, blotted, and allowed to dry flat against the slide. Staining The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections. Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylenes (or substitutes) to alcohols to water. There are no stains that can be done on .tissues containing paraffin The stained slide must go through the reverse process that it went through from paraffin section to water. The stained slide is taken through a series of alcohol solutions to remove the water, then through clearing agents to a point at which a permanent resinous substance beneath the glass cover slip. The staining process makes use of a variety of dyes that have been chosen for their ability to stain various cellular components of tissue. The routine stain is that of hematoxylin and eosin (H and E). Other stains are referred to as "special stains" because they are employed in specific situations according to the diagnostic need Frozen sections are stained by hand, because this is faster for one or a few individual sections Coverslipping

The stained section on the slide must be covered with a thin piece plastic or glass -to protect the tissue from being scratched, -to provide better optical quality for viewing under the microscope, and -to preserve the tissue section for years to come...

chemical fixatives

• There are five major groups of fixatives, classified according to mechanism of action: • Aldehydes • Mercurials • Alcohols • Oxidizing agents • Picrates • HOPE Fixative