tissue prep

• Tissue preparation


  The process of preparing tissue specimens for viewing under a microscope

  
• Steps involved in preparation of tissue

  1. Fixation

  2. Processing (dehydration, clearing, infiltration)

  3. Embedding

  4. Sectioning

  5. Mounting

  6. Staining

  
• Fixation


  The process of preserving cells and tissue components and keeping them as close to normal as possible, to allow for the preparation of thin and stained sections

  
• Fixatives


  • Disable intrinsic biomolecules, particularly proteolytic enzymes, to prevent digestion or damage of the sample
  • Protect the sample from extrinsic damage
  • Are toxic to most common microorganisms
  • Alter cells or tissues on a molecular level to increase their mechanical strength or stability
• Choice of fixative and fixation protocol

  Depends on the additional processing steps and final analyses planned
• Most commonly used fixative in histology

  Formalin (10% neutral buffered formaldehyde in water)
• Types of fixation


  • Heat fixation
  • Freezing
  • Chemical fixation
• Heat fixation


  • Preserves overall morphology but not internal structures
  • Used for fixation of single cell organisms, most commonly bacteria and archaea
• Freezing


  • Used to get rapid results but does not give fine details
  • Samples are cooled at a rate sufficient for water to be frozen in a vitreous state, without the formation of ice crystals
  • Fixation occurs in milliseconds, structures are preserved hydrated, and in as close to their native state
• Chemical fixation


  • Preserves structures in a state (both chemically and structurally) as close to living tissue as possible
  • Requires a chemical fixative that can stabilize the proteins, nucleic acids and microsubstances of the tissue by making them insoluble
• Fixative choice based on target

  • Proteins: Neutral Buffered Formalin, Paraformaldehyde
  • Enzymes: Frozen Sections
  • Lipids: Frozen Sections, Glutaraldehyde/Osmium Tetroxide
  • Nucleic Acids: Alcoholic fixatives, HOPE
  • Mucopolysaccharides: Frozen Sections
  • Biogenic Amines: Bouin Solution, Neutral Buffered Formalin
  • Glycogen: Alcoholic based fixatives
• Factors affecting fixation


  • pH of the fixative
  • Osmolarity of the fixative
  • Size of the specimen
  • Volume of the fixative
  • Temperature
  • Duration
• Tissue processing


  The process of removing water from tissues and replacing it with a medium that solidifies to allow thin sections to be cut
• Steps in tissue processing

  1. Dehydration

  2. Clearing

  3. Infiltration and impregnation
• Dehydration


  The process of transferring the tissue through a series of increasingly concentrated alcohol solutions, ending in 100%, to remove all water
• Clearing


  The process of replacing the ethanol with an organic solvent miscible with both alcohol and the embedding medium, giving the tissue a translucent appearance
• Infiltration


  The process of placing the tissue in melted paraffin until it becomes completely infiltrated with this substance
• Embedding


  The process of surrounding the tissue with a molten medium using a mold, to give support to the tissue, prevent distortion during cutting, and preserve the tissue for archival use

  
• Embedding materials


  • For light microscopy, paraffin wax is most frequently used
  • For electron microscopy, resins are the most commonly used

  
• Sectioning/Microtomy


  The process of cutting the embedded tissue into thin sections using a microtome

  
• Microtome


  • The main instrument used to cut the embedded tissue in the paraffin block into thin sections

  
• Sectioning for light microscopy

  • Steel knife mounted in a microtome is used to cut 3-5 μm thick tissue sections which are mounted on a glass microscope slide

  
• Sectioning for transmission electron microscopy

  • Diamond knife mounted in an ultra microtome is used to cut 0.1-0.5 μm thick tissue sections which are mounted on a 3-mm-diameter copper grid
• Steps after sectioning


  1. Sections are mounted onto clean glass slides and labelled

  2. Sections are floated in a water bath and then mounted on the slides

  3. Mounted sections are dried on a hot plate or a hot air oven and then kept overnight at room temperature

  4. Slides with paraffin-embedded sections can be stored either at room temperature or at 2-8 °C for several years
• SECTIONING/MICROTOMY


  1. The type of knife used

  2. The thickness of sections

  3. Vary with the type of microscope which will be used for the analysis
• MOUNTING


  • Biological tissue has little contrast under the microscope
  • The tissue section is colorless because the fixed protein has the same refractive index as that of glass
  • Staining is employed to give both contrast to the tissue as well as highlighting specific features of interest which helps us to understand the morphology of the tissue

  
• STAINING


  Dyes that have a specific affinity with the different tissue proteins and color them differently are used
• Types of dye


  • Anionic dye/acidic dye
  • Cationic dye/basic dye
  • Neutral dye
  • Ligand or Chelating dye
• Anionic dye/acidic dye


  Negatively charged, binds to cytoplasmic proteins, collagen
• Cationic dye/basic dye


  Positively charged, binds to nucleic acid, epithelial mucin
• Neutral dye


  Contains both acidic and basic dye, binds to both nucleus and cytoplasm
• Ligand or Chelating dye


  Weak acid so anionic and negatively charged, binds to nucleus and cytoplasm
• Factors that influence staining

  • Dye affinity to the target tissue specimen
  • Specimen geometry
  • Target concentration
  • Rate of reaction
  • Rate of stain loss
• Routine staining


  Hematoxylin and Eosin stain (H&E) that is routinely used with all tissue specimens to reveal the underlying tissue structures and conditions

  
• Special stains


  Alternative staining techniques that are used when H&E does not provide all the information the pathologist or researcher needs
• Common stains


  • Hematoxylin and eosin
  • Wright-Giemsa stain for blood smears
  • Silver, PAS (Periodic acid–Schiff), and acid-fast stains
• H&E stain


  • Hematoxylin stains DNA in the cell nucleus, RNA-rich portions of the cytoplasm producing a dark blue or purple color
  • Eosin stains other cytoplasmic structures and collagen pink
  • Eosin is considered a counterstain
• Blood smear staining


  • Routinely stained with mixtures of acidic (eosin) and basic (methylene blue) dyes
  • Leishman Stain is commonly used in the lab
• Leishman Stain


  • Characterized by shades of blue, pink and purple, gives a clear differentiation and understanding of cellular morphology
  • The nuclei take up a blue stain while the cytoplasm takes on a pink or purple stain depending on the type of cell and its composition
  • Allows for the differentiation of different blood cell types including RBCs, Lymphocytes, Neutrophils, Monocytes, Basophils, Platelets
  • Provides information about cellular function
• Leishman Stain Principle


  • Methylene blue stains the acidic part of the cell i.e., the nuclei (DNA) & the cytoplasm (RNA) of WBCs and granules of basophils
  • Eosin stains basic part of the cells (eosinophilic granules) and Hb of red cells
  • Methyl alcohol fixes the smear to the slide