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Preparation of histological slide

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Mohammad A. Nassir

This document discusses the process of preparing histological specimens. It involves fixing tissues to prevent degradation, processing them through dehydration, clearing and embedding in paraffin wax. Tissues are then sectioned using a microtome and stained, commonly with hematoxylin and eosin, to visualize cells and structures under a microscope. The staining highlights nucleic acids, ribosomes and other components to aid examination and study of tissue structure and organization.

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PREPARATION OF HISTOLOGICAL SPECIMENS
Histology is the study of tissues and how these tissues are arranged into organs Histo in Greek means tissue or web Tissues consist of cells and extra cellular matrix The function of the tissue depends on the interaction between cells and extracellular matrix. The small size of cells and matrix content make the study of tissues dependent on microscope and other advances in biological techniques
TISSUE PREPARATION The most widely used method of studying tissues is using histological slides. The tissue in the slide must reflect the actual nature of the tissue in the body To insure that, tissues to be studied must pass through a series of steps before examination These steps are in sequential order Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues.
TISSUE FIXATION Aim of Fixation: 1- To prevent autolysis and bacterial attack. 2- To fix the tissues so they will not change their volume and shape during processing. 3- To prepare tissue and leave it in a condition which allow clear staining of sections. 4- To leave tissue as close as their living state as possible, and no small molecules should be lost. Fixation is a reaction between the fixative and proteins in the specimen which form a gel, so keeping every thing as their in vivo relation to each
Types of Fixative Acetic acid Formaldehyde 10% Ethanol Glutaraldehyde Methanol Picric acid Osmic acid (Osmium tetroxide)
TISSUE PROCESSING Aim: Is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut , and yet soft enough not to damage the knife or tissue. Stages of processing: 1.Dehydration. 2- Clearing. 3- Embedding.
Dehydration To remove fixative and water from the tissue and replace them with dehydrating fluid. Delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol to minimize tissue distortion from diffusion currents. In the paraffin method, dehydration from aqueous fixatives is usually initiated in 60%- 70% ethanol, progressing through 90%-95% ethanol, absolute ethanol before proceeding to the clearing stage.
Types of dehydrating agents Ethanol Methanol Acetone Tissues may be held and stored indefinitely in 70% ethanol without harm
Clearing Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium. Choice of a clearing agent depends upon many factors
Types of Clearing Agents Xylene. Toluene. Chloroform. Benzene. Propylene oxide
Embedding Is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning. A substances added alone or in combination to the wax to: Improve ribboning. Increase hardness. Decrease melting point Improve adhesion between specimen and wax
Embedding Moulds A. paper boat mould B. metal boat mould C. Dimmock embedding mould D. Peel-a-way disposable mould E. Base mould used with embedding ring ( F) or cassette bases (G)
CUTTING using the microtome
A microtome is a mechanical instrument used to cut biological specimens into very thin sections for microscopic examination. Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology.
Microtome knives STEEL KNIVES NON-CORROSIVE KNIVES FOR CRYOSTATS DISPOSABLE METAL BLADES GLASS KNIVES DIAMOND KNIVES
STAINING
Hematoxylin and Eosin (H & E) H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections.
Staining Procedure Deparaffinize and hydrate to water If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate (hypo) Mayer's hematoxylin for 15 minutes Counterstain with eosin from 15 seconds to 2 minutes depending on the age of the eosin, and the depth of the counterstain desired Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess eosin is removed Clear in xylene, two changes of 2 minutes each Mount in Permount or Histoclad
Staining machine
Basic dyes stain: Heterochromatin Nucleic acids Ribosomes Cartilage Acidic dyes stain: Filaments Mitochondria Collagen Muscle fibers
Additional Dyes Many tissue components can not be stained with (Hematoxylene and Eosin). Other dyes are used to specifically stain certain tissue components Resorcin-Fuchsin for elastic fibers Silver stain for reticular fibers and basement membrane Periodic-Acid Schiff (PAS) Reaction for CHO
Preparation of histological slide
Preparation of histological slide

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Preparation of histological slide

  • 2. Histology is the study of tissues and how these tissues are arranged into organs Histo in Greek means tissue or web Tissues consist of cells and extra cellular matrix The function of the tissue depends on the interaction between cells and extracellular matrix. The small size of cells and matrix content make the study of tissues dependent on microscope and other advances in biological techniques
  • 3. TISSUE PREPARATION The most widely used method of studying tissues is using histological slides. The tissue in the slide must reflect the actual nature of the tissue in the body To insure that, tissues to be studied must pass through a series of steps before examination These steps are in sequential order Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues.
  • 4. TISSUE FIXATION Aim of Fixation: 1- To prevent autolysis and bacterial attack. 2- To fix the tissues so they will not change their volume and shape during processing. 3- To prepare tissue and leave it in a condition which allow clear staining of sections. 4- To leave tissue as close as their living state as possible, and no small molecules should be lost. Fixation is a reaction between the fixative and proteins in the specimen which form a gel, so keeping every thing as their in vivo relation to each
  • 5. Types of Fixative Acetic acid Formaldehyde 10% Ethanol Glutaraldehyde Methanol Picric acid Osmic acid (Osmium tetroxide)
  • 6. TISSUE PROCESSING Aim: Is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut , and yet soft enough not to damage the knife or tissue. Stages of processing: 1.Dehydration. 2- Clearing. 3- Embedding.
  • 7. Dehydration To remove fixative and water from the tissue and replace them with dehydrating fluid. Delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol to minimize tissue distortion from diffusion currents. In the paraffin method, dehydration from aqueous fixatives is usually initiated in 60%- 70% ethanol, progressing through 90%-95% ethanol, absolute ethanol before proceeding to the clearing stage.
  • 8. Types of dehydrating agents Ethanol Methanol Acetone Tissues may be held and stored indefinitely in 70% ethanol without harm
  • 9. Clearing Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium. Choice of a clearing agent depends upon many factors
  • 10. Types of Clearing Agents Xylene. Toluene. Chloroform. Benzene. Propylene oxide
  • 11. Embedding Is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning. A substances added alone or in combination to the wax to: Improve ribboning. Increase hardness. Decrease melting point Improve adhesion between specimen and wax
  • 12. Embedding Moulds A. paper boat mould B. metal boat mould C. Dimmock embedding mould D. Peel-a-way disposable mould E. Base mould used with embedding ring ( F) or cassette bases (G)
  • 13. CUTTING using the microtome
  • 14. A microtome is a mechanical instrument used to cut biological specimens into very thin sections for microscopic examination. Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology.
  • 15. Microtome knives STEEL KNIVES NON-CORROSIVE KNIVES FOR CRYOSTATS DISPOSABLE METAL BLADES GLASS KNIVES DIAMOND KNIVES
  • 17. Hematoxylin and Eosin (H & E) H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections.
  • 18. Staining Procedure Deparaffinize and hydrate to water If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate (hypo) Mayer's hematoxylin for 15 minutes Counterstain with eosin from 15 seconds to 2 minutes depending on the age of the eosin, and the depth of the counterstain desired Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess eosin is removed Clear in xylene, two changes of 2 minutes each Mount in Permount or Histoclad
  • 20. Basic dyes stain: Heterochromatin Nucleic acids Ribosomes Cartilage Acidic dyes stain: Filaments Mitochondria Collagen Muscle fibers
  • 21. Additional Dyes Many tissue components can not be stained with (Hematoxylene and Eosin). Other dyes are used to specifically stain certain tissue components Resorcin-Fuchsin for elastic fibers Silver stain for reticular fibers and basement membrane Periodic-Acid Schiff (PAS) Reaction for CHO