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INTRODUCTION:- The gradual development of immunohistochemical methodologies over the pastdecades has allowed the identification of specific or highly selective cellularepitopes in formalin-fixed paraffin-embedded tissues with an antibody and appropriate labeling system  IMMUNOHISTOCHEMISTRY :- Immunohistochemistry is a technique for identifying cellular or tissue constituents (antigens) by means of antigen-antibody interactions, the site of binding can be identified by direct labeling of the antibody or by use of a secondary labeling method. Antigen-Antibody binding :- The amino acid side-chains of the variable domain of an antibody form a cavity which is complementary to a single type of antigen like a lock and key. The  precise fit required explains the high degree of specificity seen in antigen antibody interaction. Affinity:- is the 3 dimensional fit of the antibody to its specific antigen and is a measure of the binding strength between antigen and antibody. Avidity: is the functional combined strength of an antibody with its antigen. An antibody against more than one epitope of an antigen will bind more strongly to it. Antibody specificity: is the characteristic of an antibody to bind selectively to a single epitope or an antigen. Sensitivity: is the relative amount of an antigen that a technique is able to detect. Primary reagents :- Polyclonal antibodies: they are produced by immunizing an animal with a purified specific molecule (immunogen) bearing the antigen of interest. The animal will mount a humoral response to the immunogen and the antibodies so produced can be harvested from animal blood. The serum is polyclonal in nature as it comprises of a mixture of antibodies to different epitopes present on the antigen. Some of these antibodies may cross react with other molecules and produce nonspecific staining. Monoclonal antibodies:-  Hybridoma method is used to produce these antibodies and it combines the ability of a plasma cells or transformed B lymphocytes to produce a specific antibody with the in vitro immortality of a neoplastic myeloma cell line. With the technique of cloning, this cell can be grown and multiplied in cell culture to unlimited numbers and can produce large supply of particular antibodies. Labels:-  Enzymes are the most widely used labels in IHC, and incubation with a chromogen using a standard histochemical method produces a stable, colored reaction end-product suitable for the light microscope. Horseradish peroxidase is the most widely used enzyme, and in combination with the most favored  chromogen, i.e. 3,3’- diaminobenzidine tetrahydrochloride (DAB) it gives a crisp, insoluble, stable, dark brown reaction end-product. Immunohistochemical Methods:- Methods There are numerous IHC staining techniques that may be used, the selection should be based on parameters such as type of specimen, type of preparation (frozen or paraffin section) and sensitivity required. Traditional Direct technique: the primary antibody is conjugated directly to the label. The conjugate may be either a fluorochrome or enzyme. The labeled antibody reacts directly with the antigen. The technique is quick and easy to use but provides little signal amplification and is less sensitive, so its used to demonstrate immunoglobulins and complements in frozen sections of skin and renal biopsies. New direct technique (Enhanced polymer one step staining method): available under the commercial name of EPOS. A large number of primary antibodies and peroxidase enzymes are attached to a dextran polymer ‘backbone’, hence increasing the signal amplification. Two step indirect technique: A labeled secondary antibody directed against the immunoglobulin of the animal species in which the primary antibody has been raised visualizes an unlabeled primary antibody. It is more sensitive than direct technique. Antigen retrieval :- The demonstration of many antigens can be significantly improved by thepretreatment with the antigen retrieval reagents that break the protein cross-linksformed by formalin fixation and thereby uncover hidden antigenic sites. It canbe done by enzymatic method and/or heat induced. The most popular enzymes employed today are trypsin and protease. The enzymatic digestion breaks down formalin cross-linking and hence the antigenic sites are uncovered. Heat based antigen retrieval methods have brought great improvement in IHC. The theories suggested for the role of heat pretreatment include: heavy salts act as protein precipitant forming insoluble complexes with polypeptides. Another theory is that heat mediated retrieval removes the weaker Schiff bases formed during formalin fixation. The different methods of heat based antigen retrieval include 1. Microwave antigen retrieval 2. Pressure cooker antigen retrieval 3. Steamer 4. Water bath Microwave antigen retrieval with a non toxic citrate buffer at pH 6.0 has demonstrated results equivalent to frozen sections. Most domestic microwave ovens are suitable for antigen retrieval . Uneven heating and the production of hot-spots have been reported, but using 400-600 ml of buffer in a suitably sized container can minimize these problems. Pressure cooker has been suggested as an alternative to microwave oven. Batch variation and production of hot and cold spots in microwave can be overcome. Pressure cooker is said to be more uniform in heating. Also the increased temperature (120°C) attained under pressure is an advantage in unmasking antigens. Buffers used for antigen retrieval: *Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0) *Tri-sodium citrate (dihydrate) 2.94 g *Distilled water 1000 ml *Mix to dissolve. Adjust pH to 6.0 with 1N HCl. *Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage. *1 mM EDTA, adjusted to pH 8.0 *EDTA 0.37 g *Distilled water 1000 ml *Store at room temperature for 3 months *Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween *20, pH 9.0) *Tris 1.21 g *EDTA 0.37 g *Distilled water 1000 ml (100 ml to make 10x, 50 ml to make 20x) *Mix to dissolve. pH is usually at 9.0. *Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage IHC staining All incubations should be carried out in a humidified chamber to avoid drying of the tissue. Drying at any stage will lead to non-specific binding and ultimately high background staining. A shallow, plastic box with a sealed lid and wet tissue paper in the bottom is an adequate chamber,

INTRODUCTION:- Sections are prepared quickly for histological examination by freezing the tissue. The section should be thin, and without water crystals. It is an important procedure for quick diagnosis. OBJECTIVES:- After reading this lesson, you will be able to: *enlist the indications of frozen section *explain the disadvantages of frozen section *describe cryostat PURPOSES OF FROZEN SECTION :- Frozen sections are used for following purpose: *Quick diagnosis *Study the margins of cancer *Enzyme histochemistry *Immunohistochemistry *Detection of lipid *Some molecular procedures Disadvantages:-  *Morphology is distorted *Cellular details are not well seen, *Staining is not very good *Some specials stains cannot be performed. Handling of specimen:- Tissue must reach histopathology laboratory immediately. To avoid tissue being dried it should be kept in saline. The size of the tissue should be small thin, so that good smooth sections can be obtained and freezing is quick. Thickness of he tissue should be about 3mm to 4mm.The tissue can directly be taken to cryostat or can be fixed with 10% formalin or formol –alcohol Embedding media:- Sucrose (20%) or a drop of water may be applied on the chuck. Optimum Cooling temperature (OCT) compounds or 20% sucrose gives good result. Other embedding media are available with cryostat. Completion of freezing is observed by the change of color of tissue which turns glossy white. Freezing should be done fast. This will prevent ice crystal formation. The morphology is better preserved and artifacts are lesDifferent freezing substances are used depending upon the availability and feasibility. Carbon Dioxide gas is most commonly used with freezing microtome. This gives good results. Liquid Nitrogen is another substance used for freezing the tissue. An expertise is required while using liquid nitrogen to get uniform freezing. Aerosol sprays are also used for this purpose Cryostat: Cryostat is used in medicine to cut histological sections. They are usually used in a process called frozen section histology. The cryostat is essentially an ultrafine “deli-slicer”, called a microtome, placed in a freezer. The cryostat is usually a stationary upright freezer, with an external wheel for rotating the microtome. The temperature can be varied, depending on the tissue being cut – usually from minus 20 to minus 30 degree Celsius. The freezer is either powered by electricity, or by a refrigerant like liquid nitrogen. Small portable cryostats are available and can run off generators or vehicle inverters. To minimize unnecessary warming all necessary mechanical movements of the microtome can be achieved by hand via a wheel mounted outside the chamber. Newer microtomes have electric push button advancement of the tissue. The precision of the cutting is in micrometres. Tissue are sectioned as thin as 1 micrometre. Usual histology slides are mounted with a thickness of about 7 micrometres. Specimens that are soft at room temperature are mounted on a cutting medium (often made of egg white) on a metal “chuck”, and frozen to cutting temperature (for example at -20 degrees C). Once frozen, the specimen on the chuck is mounted on the microtome. The crank is rotated and the specimen advances toward the cutting blade. Once the specimen is cut to a satisfactory quality, it is mounted on a warm (room temperature) clear glass slide, where it will instantaneously melt and adhere. The glass slide and specimen are air dried, and stained. The entire process from mounting to reading the slide takes from 10 to 20 minutes, allowing rapid diagnosis in the operating room, for the surgical excision of cancer. The cryostat section quality is poorer as compared to fixed tissue sections    

INTRODUCTION:- The sections, as they are prepared, are colourless and different components cannot be appreciated. Staining them by different coloured dyes, having affinities of specific components of tissues, makes identification and study of their morphology possible. Hematoxylin and Eosin (H&E) is the most frequently used stain in histology. OBJECTIVES:- After reading this lesson, you will be able to: describe Hematoxylin and its preparation describe the properties of Hematoxylin explain Eosin and its preparation describe the method of staining. HEMATOXYLIN :- It is extracted from the bark of a tree”, hematoxylom campechianum”. The hematoxylin which we buy is extracted from this bloodwood tree. To obtain the bark of freshly logged tree is chipped off, then boil the chips in water. An orange red solution is obtained, which turns yellow, then black on cooling. The water is evaporated leaving crude hematoxylin. Further purification is done.Solutions of the dye should be oxidized to retain its staining ability longer. The dye may be oxidized by exposure to the natural light for 3-4 months. chemical oxidation is achieved by using either sodium iodate or mercuric oxide. The chemical oxidation converts the dye almost instantaneously but the product does not have shelf life. Sodium iodate is most commonly used oxidizing agent (0.2gm oxidizes 1.0 gm hematoxylin). Hematoxylin is neither a dye nor it has coloring properties. For nuclear staining it is necessary to oxidize the hematoxylin to hematin which is a weak anionic purple dye. Anionic hematin will have no affinity for the nucleic acids of nuclei. Hence a metallic salt or mordant is combined with hematoxylin so that a positive charge to the dye is obtained by virtue of the metal action. Thus the cationic dye–metal complex will bind to the anionic nuclear chromatin. Various mordants are ammonium or potassium alum ferric salt, chrom alum and phosphotungstic acid. The tissue component most frequently demonstrated is nuclear chromatin using an alum mordant in the H&E staining method. The combination of hematoxylin and mordant is called a hematoxylin lake. The aluminium lake formed with ammonium alum is particularly useful for staining nuclei. Hematoxylin recipes using these mordants are called alum hematoxylin. PROPERTIES OF HEMATOXYLIN :- 1. Hematoxylin has no staining property. 2. Hematin with mordant such as ammonium or potassium alum forms lake which functions as cationic dye and stains anionic tissue components. 3. Hematin in an aqueous solution can be acidic or an alkaline dye depending on pH. 4. Hematin has affinity for several tissues with an appropriate mordant. Progessive staining :-When tissue is left in the stain just long enough to reach the proper end point. The slides have to be examined at different interval to find out when the staining is optimum. Regressive staining :-  In this method the tissue is overstained and then destained (differentiate) until the proper endpoint is reached. Harris hematoxylin is a regressive stain; the overstaining is removed by acid alcohol. The removal of this excess dye is called differentiation. The hematoxylin alum gives a reddish hue to the tissues because of acidic pH. To convert this colour to the final blue, alkaline pH is required. This process is called “blueing”. It is done either by tap water or by ammonium hydroxide. Preparation of Harris’s hematoxylin :- Ingredients :-  Hematoxylin                             –          5 gm Absolute alcohol                     –          50 ml Ammonium alum                    –         100 gm Distilled water                         –         1000 ml Mercuric oxide                        –         2.5 gm Glacial acetic acid  5gm        –         40 ml Method :-  Dissolve the hematoxylin in absolute alcohol and ammonium alum in hot water. Mix the two solutions and heat to boiling. Remove from flame, and add mercuric oxide and cool rapidly. Glacial acetic acid if added gives brisk nuclear staining, but life of the solution is reduced. Hence if acetic acid is to be added, it should be added in working solution. Preparation of Mayer’s hematoxylin:- Ingredients : Hematoxylin                             –   1.0 gm                                                                                                                    Distilled water                         –    1000 ml                                                                                                                  Ammonium alum                   –     50 gm Sodium iodate                        –      0.2 gm Citric acid (reduces pH)        –     1.0 gm Chloral hydrate (preservative)-   50 gm Method:- Hematoxylin is dissolved in distilled water using gentle heat. Then alum is added and dissolved. Then sodium iodate, citric acid and chloral hydrate are added respectively. EOSIN :- Eosin is used as the counterstain that stains the cytoplasm rose coloured. The intensity of the eosin is individual choice. The most widely used eosin is “eosin Y”. The “Y” stands for yellowish. It is available in either water soluble or alcohol soluble form. Most laboratories use the water soluble form of eosin Y in an alcohol-water solution which is described here. Eosin Y (water soluble)          –         1.0 gm Distilled water 

INTRODUCTION:- Embedding is the process in which the tissues or the specimens are enclosed ina mass of the embedding medium using a mould. Since the tissue blocks are very thin in thickness they need a supporting medium in which the tissue blocks are embedded. This supporting medium is called embedding medium. Various embedding substances are paraffin wax, celloidin, synthetic resins, gelatine, etc. OBJECTIVES:- After reading this lesson, you will be able to: describe embedding *explain embedding media *describe types of moulds *explain various devices for tissue embedding. EMBEDDING:- The choice of embedding media depends upon *Type of microscope *Type of microtome *Type of tissue eg. hard tissue like bone or soft tissue like liver biopsy Paraffin wax: with a higher melting point (56 to 62oC) is used for embedding.The molten wax is filtered inside the oven through a course filter paper into another container. This will protect the knife edge. OTHER TYPES OF EMBEDDING MEDIA:- Carbowax: It is a water soluble wax. Therefore tissues are directly transferred to water soluble wax after fixation and washing. Methacrylate: It is easily miscible with alcohol and gives a clear and hard block when polymerised Polymerization takes place in the presence of a catalyst. Any trace of water causes uneven polymerization and formation of bubbles in the block around the tissue Epoxy Resin (Araldite): Epoxy polymers of araldite is used in higher resolution work and to see greater details. Epoxy resins are used for electron microscopy. Epoxy polymers of araldite differ from methcrylate in that they are crosslinked causing the cured solid block of araldite to be insoluble in any solvent. Longer filtration is required because the viscosity of resin is greater than methacrylate. For electron microscopy araldite is obtained as casting resinCY212, ahardner DDSA and an amine accelerator, DMP (ditrimethylamino methyl phenol) Blocks are suitably cured before sectioning for 48 to 60 hours at 60oC Agar embedding: It is mainly used in double embedding. Multiple fragments and friable tissue may be impregnated in one block when sectioning on the cryostat. Another use of agar embedding is for FNAC specimens. Celloidin media:- Celloidin is a purified form of nitrocellulose. It is used for cutting hard tissues. Gelatin: Its melting point is less than the melting point of agar. Gelatin maybe used when frozen sections are required on friable and necrotic tissues. TYPES OF MOULDS  A variety of moulds are used for embedding. These may be LEUCKHARD embedding moulds (L mould) paper blocks, plastic moulds. Most of the laboratories use L moulds. L moulds are made up of metal, easy to procure, reusable and may be adjusted to make different size of blocks. One limb of the”L” is longer than the other. The two “Ls” are jointed to form a sides of the rectangular box that act as a cast to make the mould. Plastic moulds: Most of the laboratories use plastic embedding rings now. These are relatively inexpensive, convenient and support the block during sectioning and are designed to fit it on the microtome. This eliminates the step of mounting or attaching the block on a holder (metal or wooden holder). 1. Tissue-Tek System1 or Mark1 system:- In this system plastic embedding rings with stainless steel moulds allow rapid embedding and cutting of tissues. In this system the blocks are stored with the plastic rings; the angle does not change for further requirement of sections. The disadvantage of this method is that the space required for storing is more. 2. Tissue-Tek system 2or Mark 2 system: The Mark 2 system has provided a cassette to hold tissue during processing and has a stainless steel lid on the plastic cassette. The cassette has a rough surface on one side of it with a slope where the accession number or the marking is done using a permanent marker. 2. Tissue-Tek system 2or Mark 2 system: The Mark 2 system has provided a cassette to hold tissue during processing and has a stainless steel lid on the plastic cassette. The cassette has a rough surface on one side of it with a slope where the accession number or the marking is done using a permanent marker. Advantages :-  *Since the cassette is processed with the tissues and afterwards used for embedding, the writing has to be done once. *Cassettes are thin so less wax is required. *The space required for filing the blocks is less. Disadvantages:- *A special clamp has to be used in the microtome for this technique. *The cassettes are shallow hence thin sections should be taken for processing. PARAFFIN WAX ADDITIVES:- Various substances can be added to paraffin wax in order to modify its consistency and melting point to improve the efficiency during microscopy. Additives increase the hardness of blocks. This helps in cutting thinner sections at higher temperature. Stickiness of the medium is increased so better ribbons can be obtained. However if larger quantities of additives are added, undesirable side effects may be seen. Commonly used additives:- Ceresin – It is hard white substance derived from mineral ozokerite. Its melting point is between 61 to70o C. The addition of 0.3-0.5% is sufficient to reduce the crystalline structure of paraffin wax. Bees’ wax – It is yellow substance with melting point of 64o C. This also reduces the crystalline structure of the paraffin wax and improves the ribbon quality. Bayberry wax – It is a vegetable wax and present in the peal of bayberry. It is extracted from the peal of the fruit. Its melting point is 45° C. Devices for tissue embedding :- Devices designed specifically for tissue embedding are available for laboratories in need of such equipment. These machines vary in size and design depending on the number of samples they are designed to process. Some are designed for specific embedding media, including proprietary compounds intended for specific kinds of histopathology applications. Tissue embedding equipment tends to be expensive. Manufacturers have sales representatives who can provide information and advice when a lab is selecting new or replacement Tissue embedding machine equipment. All the blocking steps can be performed with the help of tissue embedding machine. The embedding machine