Histological Techniques for Tissue Preparation

INTRODUCTION Histological technique deals with the preparation of tissue Histological technique deals with the preparation of tissue for microscopic examination. for microscopic examination. The aim of good histological technique to preserve The aim of good histological technique to preserve microscopic anatomy of tissue. microscopic anatomy of tissue. Make them hard so that very thin section ( Make them hard so that very thin section (4 4 to can be made. can be made. Good staining should be possible. Good staining should be possible. to 5 5 micron) micron) After staining, After staining, the section should represent the anatomy of the section should represent the anatomy of the tissue as close to as possible to their structure in the tissue as close to as possible to their structure in life. life. This is achieved by passing the total as selected part of This is achieved by passing the total as selected part of the tissue through a series of process. the tissue through a series of process.

TECHNIQUES These techniques are: These techniques are: 1. Fixation 1. Fixation 2. Dehydration 2. Dehydration 3. Cleaning 3. Cleaning 4. Embedding 4. Embedding 5. Cutting 5. Cutting 6. Staining 6. Staining

FIXATION FIXATION This is the process by which cells and This is the process by which cells and tissue are fixed in a physical and chemical tissue are fixed in a physical and chemical state so that they will withstand subsequent state so that they will withstand subsequent treatment with various reagents with treatment with various reagents with minimum loss of architecture minimum loss of architecture . . This should be approximately 10 This should be approximately 10- -20 times the volume of the specimen. Fixative should volume of the specimen. Fixative should surround the specimen on all sides. surround the specimen on all sides. Histochemical fixatives :Formal Histochemical fixatives :Formal saline,Cold acetone ,Absolute alcohol acetone ,Absolute alcohol 20 times the saline,Cold

PROPERTIES OF AN IDEAL FIXATIVE Prevents autolysis and bacterial decomposition. Prevents autolysis and bacterial decomposition. Preserves tissue in their natural state and fix all Preserves tissue in their natural state and fix all components. components. Make the cellular components insoluble to reagent used Make the cellular components insoluble to reagent used in tissue processing. in tissue processing. Preserves tissue volume. Preserves tissue volume.

SEQUENCE OF TISSUE PROCESSING SEQUENCE OF TISSUE PROCESSING Dehydration Dehydration: :Tissues increasing strength of alcohol; e.g. increasing strength of alcohol; e.g. 50 and and 100 100%. %. Clearing: Clearing:During During dehydration water in tissue has been dehydration water in tissue has been replaced by alcohol. The next step alcohol should be replaced by alcohol. The next step alcohol should be replaced by paraffin wax. replaced by paraffin wax. Clearing of tissue is achieved by any of the following Clearing of tissue is achieved by any of the following reagents:Xylene reagents:Xylene, , Chloroform,Benzene Chloroform,Benzene Note: Note:Xylene Xylene is commonly used. is commonly used. Impregnation with Wax Impregnation with Wax This is allowed to occur at melting point temperature of This is allowed to occur at melting point temperature of paraffin wax, which is paraffin wax, which is 54 54- -60 should be about should be about 25 25- -30 30 times the volume of tissues. times the volume of tissues. . . Tissues are dehydrated by using are dehydrated by using 50%, %, 70 70%, %, 90 90% % 60oC. Volume of wax oC. Volume of wax

Embedding Embedding Impregnated tissues are placed in a Impregnated tissues are placed in a mould and then fresh melted wax is poured in it and allowed to and then fresh melted wax is poured in it and allowed to settle and solidify. settle and solidify. Once the block has cooled sufficiently to form a surface Once the block has cooled sufficiently to form a surface skin it should be immersed in cold water to cool it rapidly. skin it should be immersed in cold water to cool it rapidly. . . Microtomy Microtomy For For light microscopy light microscopy emotorcim a ni detnuom efink ssalg a , emotorcim a ni detnuom efink ssalg a , tuc ot desu si tuc ot desu si4 4- -6 6 um um- -thick tissue sections which are thick tissue sections which are mounted on a glass microscope slide mounted on a glass microscope slide . mould with their labels with their labels

STAINING STAINING Staining is a process by which we give color to a section. Staining is a process by which we give color to a section. There are hundreds of stains available. There are hundreds of stains available. The stains can be hematoxylin/eosin stain or special stains The hematoxylin/eosin stain is usually abbreviated as h&e stain. The H&E stain is routinely used. It gives the nucleus a blue color & the cytoplasm & the extracellular matrix a pinkish color.

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