Tissue Processing, Staining, and Mounting Media PDF

Tissue processing, Staining, Adhesives and Mounting Media Important stages to be discuss o Fixation o Dehydration o Clearing (de alcoholization) o Infiltration/impregnation Staining 3 MAJOR GROUPS OF STAINING HISTOLOGICAL STAINING HISTOCHEMICAL STAINING IMMUHISTOCHEMICAL STAINING...

Tissue processing, Staining, Adhesives and Mounting Media Important stages to be discuss o Fixation o Dehydration o Clearing (de alcoholization) o Infiltration/impregnation Staining 3 MAJOR GROUPS OF STAINING HISTOLOGICAL STAINING HISTOCHEMICAL STAINING IMMUHISTOCHEMICAL STAINING HISTOLOGICAL STAINING HISTOCHEMICAL STAINING IMMUNOHISTOCHEMICAL STAINING - Is a combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated under the microscope. - Uses a wide range of monoclonal or polyclonal, fluorescent-labeled or enzyme-labeled antibodies. METHODS 1. Direct staining 2. Indirect staining 3. Progressive staining 4. Regressive staining 5. Metachromatic staining 6. Counterstaining 7. Metallic impregnation DIRECT STAINING INDIRECT STAINING PROGRESSIVE STAINING REGRESSIVE STAINING DIFFERENTIATION Selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues. Done by washing the section in simple solution, or by the use of acids and oxidizing agents. ALCOHOL –act as differentiator for both basic and acidic dyes simply dissolving out the excess dye. A mordant can act as a differentiating agent Usually controlled by following the exact times specified for staining, or by examination under the microscope. COUNTER STAINING VITAL STAINING - Is the selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle - Nucleus of the living cell is resistant to vital stain - INTRAVITAL STAINING –done by injecting the dye into any part of the animal body producing specific coloration of certain cells. > Common dyes used are lithium, carmine, and India ink. - SUPRAVITAL STAINING –is used to stain living cells immediately after removal from the living body. - Thin slices of tissues are placed in small staining dishes and enough staining solution is added to cover the tissue.s. MOUNTING MEDIUM –Usually a syrupy fluid applied between the section and the coverslip –Protect the stained section from getting scratched –Protect the stained section from bleaching or deterioration due to oxidation –Facilitate easy handling and storage –Prevent distortion of image during microscopic examination –Excess xylene is dried off from the section a drop of mounting medium is placed at the center of the slide, a dry cover slip is placed, incubate the slide at 37°C for 12-24 hours –If excess xylene is not removed à bubbles –If sections are to be remounted à covered glass can be removed by soaking in xylene CHARACTERISTICS OF A GOOD MOUNTING MEDIUM: –Refractive index of the mountant should be as near as possible to that of the glass which is 1.518 –Should be freely miscible w/ xylene and toluene –Should not dry quickly –Should not crack or produce artefactual granularity on the slide upon drying –Should not dissolve out or fade tissue sections –Should not cause shrinkage and distortion of tissues –Should not leach out any stain or affect staining–Should not change in color or pH –Should set hard, thereby producing permanent mounting of sections AQUEOUS MOUNTING MEDIA –Designed to mount water-miscible preparations directly from water in cases where the stain is removed or decolorized w/ alcohol or xylene –Usually made up of gelatin, glycerin jelly or gum arabic (solidify the medium), glycerol (prevent cracking or drying of the preparation), sugar ( increase refractive index) and a preservative solution WATER –Low refractive index, moderately transparent and evaporates quickly –Good for temporary mounting –Does not allow tissue to be examine under oil immersion lens GLYCERIN MOUNTING MEDIUM