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Submitted By, Vikash kumar Roll no RZ1051A09 Registration no 11004789 Subject Histotechnology-1 Subject code M.L.T 217 Section Rz1051A

Submitted To, Mr.Harpreet Singh Deptt - Paramedical Science

Introduction of Embedding Tissue embedding is a tissue preparation technique used when a technician needs to be able to cut very small samples of tissue with a device known as a microtome. The tissue is supported in a medium, allowing the technician to create even, accurate cuts without crushing or otherwise damaging the tissue. The slices of tissue can be examined under a microscope to study their characteristics and collect information for use in diagnostic evaluations and other studies. Samples of tissue can come from surgical biopsies, autopsies, and many other sources. The technician places the tissue into a cassette designed to be submerged in the embeddingmedium. Paraffin, plastics, epoxies, and some types of gel media can all be used. Once the medium has set, the sample can be pulled back out, inspected to confirm the embedding went smoothly, and sliced as desired. Thanks to the support of the medium, very fine tissue slices are available, allowing the technician to visualize a high level of detail. Placement of samples in the cassette requires some training and experience, as the tissueneeds to positioned appropriately for the type of study being conducted. If it is not placed right, it will be difficult to cut the desired cross sections and the sample may be ruined. Care must also be taken to process samples before embedding, Devices designed specifically for tissue embedding are available for labs 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. Sliced sections of tissue can be mounted on slides and examined or put in storage. If part of a sample is left intact, it will also be stored after tissue embedding in case additional tests are needed in the future. Stored specimens can also be used for scientific research. The sliced samples can be stained to highlight certain features or to look for traces of specific chemicals and other compounds After the tissues have been dehydrated, cleared, and infiltrated with the embedding material, they are ready for external embedding. During this process the tissue samples are placed into molds along with liquid embedding material (such as agar, gelatine, or wax) which is then hardened. This is achieved by cooling in the case of paraffin wax and heating (curing) in the case of the epoxy resins. The acrylic resins are polymerised by heat, ultraviolet light, or chemical catalysts. The hardened blocks containing the tissue samples are then ready to be sectioned. Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, FFPE tissues are an important resource for historical studies in medicine.

Embedding can also be accomplished using frozen, non-fixed tissue in a water-based medium. Pre-frozen tissues are placed into molds with the liquid embedding material, usually a water-based glycol, OCT, TBS, Cryogel, or resin, which is then frozen to form hardened blocks. Embedding and Microtomy Once the tissue has been processed it is ready to be orientated into a paraffin block and subsequently sectioned. Orientation during embedding is crucial for the representation of proper morphology. Structures in skin, small gastrointestinal biopsies, and vas deferens are among those for which orientation is especially critical. Good microtomy techniques will minimize artifacts that lead to difficult diagnostic interpretation of special stains. One of the most directly correlated factors is the thickness in which a specimen is cut. Routine H&E stained specimens are cut 34 m, but some morphology is best represented otherwise. For example, amyloid deposits are better represented at 812 m, whereas kidney biopsies should be cut at 2 m for optimal viewing of the structures of glomeruli. Techniques often used to aid in microtomy are water bath adhesive and positively charged slides. However, in some silver impregnation stains, the silver ions are attracted to the coating and produce an overall background to the slide. To avoid this artifact (Figure 3), use clean slides without a coating. After sectioning, the tissue slide is drained and may be gently heated to evaporate the layer of water between the sections and the glass. When all the water is gone, it is permissible to heat the slide enough to melt the wax, a procedure that may improve adhesion. For optimal melting of paraffin consult the melting temperature and plastic point on the manufacturing product insert. The plastic point is usually a few degrees lower than the melting point and represents the lowest temperature at which permanent deformation can occur without fracture. Types of embedding 1.Paraffin wax 2. Plastics embedding 3. Epoxies embedding 4.Plaster embedding 5.Gel embedding 6. Paraffin wax Paraffin wax a typical melting point between about 46 and 68 C (115 and 154 F), and having a density of around 0.9 g/cm It is insoluble in water, but soluble in ether, benzene, and certain esters. Paraffin is unaffected by most common chemical reagents but burns readily.

Embedding tissues in paraffin wax:Tissues are embedded by placing them in a mold filled with melted embedding medium which is then allowed to solidify. Embedding requirements and procedures are essentially the same for all waxes, and only the technique for paraffin wax is provided here in detail. At the completion of processing, tissues are held in clean paraffin wax which is free of solvent and particulate matter. Requirements for embedding are as follows: a supply of clean, filtered paraffin wax held at 2-4C above its melting point. a cold plate to rapidly cool the wax. a supply of molds in which to embed the tissues. These elements are conveniently combined in commercially available embedding stations. Otherwise a wax dispenser, embedding oven and ice will suffice. There are four main mold systems and associated embedding protocols presently in use: traditional methods using paper boats; Leuckart or Dimmock embedding irons or metal containers; the Peel-a-way system using disposable plastic molds and; systems using embedding rings or cassettebases which become an integral part of the block and serve as the block holder in the microtome. General Embedding Procedure 1. Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. 2. Select the mould, there should be sufficient room for the tissue with allowance for at leasta 2 mm surrounding margin of wax. 3. Fill the mould with paraffin wax. 4. Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time. 5. Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. 6. Insert the identifying label or place the labeled embedding ring or cassette base onto the mould.

7. Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface. 8. Remove the block from the mould. 9. Cross check block, label and worksheet. Alternative embedding media and processing methods Alternative embedding media may provide optimum support for tissues in applications for which paraffin waxes are unsuited, for example when: tissue components are heat or reagent labile hard or dense tissues are inadequately supported adhesion between specimen and wax is poor very thick or very thin sections are required sectioning whole organs such as lung or brain. Resin embedding methods are now used for many of these applications. Non-resinous embedding media include those listed below. AQUEOUS MEDIA Agar has a high melting point and low gelling temperature of agar make it ideal for double embedding multiple small tissue fragments. Agar is generally unstained by overnight stains, but will stain with alcian blue. Gelatin is used for simple embedding in a similar manner to agar. However the low melting point of gelatin (35-40C) makes it unsuitable for double embedding. It is used in Gough and Wentworth's whole-organ sectioning method and its variants, or simply to support large tissue blocks for 1 mm thick sectioning and subsequent three-dimensional reconstruction. In phospholipid and enzyme studies tissues may be infiltrated and embedded in gelatin and the resulting blocks sectioned on a freezing microtome. This technique has now largely been superseded by other media used for cryotomy. Importance of Paraffine Wax Embedding Where the best possible morphology is required, animals should be anesthesized and subjected to cardiac perfusion with saline, followed by a 10% formalin flush. If biochemical studies need to be performed on the tissue, a 10% formalin flush should not be used as it may interfere with subsequent analysis.

1. For routine stains where perfusion is not required, tissue is sectioned and drop-fixed in a 10% formalin solution. Fixative volume should be 20 times that of tissue on a weight per volume; use 2 ml of formalin per 100 mg of tissue. 2. Due to the slow rate of diffusion of formalin (0.5 mm hr), tissue should be sectioned into 3 mm slices on cooled brain before transfer into formalin. This will ensure the best possible preservation of tissue and offers rapid uniform penetration and fixation of tissue within 3 hours. 3. Tissue should be fixed for a minimum 48 hours at room temperature. 4. After 48 hours of fixation, move tissue into 70% ethanol for long term storage. 5. Keep fixation conditions standard for a particular study in order to minimize variability. (Although set times are best, tissue may be fixed for substantially longer periods without apparent harm.

Refrences I. II. III. IV. http://www.wisegeek.com/what-is-tissue-embedding.htm http://en.wikipedia.org/wiki/Paraffin http://www.histologycourse.com/Embedding%20Techniques.pdf http://protocolsonline.com/histology/paraffin-processing-of-tissue/