Tissue preparation

• Preparation of tissues for study

  1. Fixation

  2. Dehydration

  3. Clearing

  4. Infiltration

  5. Embedding

  6. Trimming
• Embedding involves placing the tissue into a solidifying medium (e.g., paraffin wax) to hold its shape during sectioning.
• Fixation


  Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, which preserve cell and tissue structure
• Preparation of tissues for study

  1. Fixation

  2. Dehydration

  3. Clearing

  4. Infiltration

  5. Embedding

  6. Trimming
• Formalin


  Buffered isotonic solution of 37% formaldehyde, a widely used fixative for light microscopy
• Glutaraldehyde


  A fixative used for electron microscopy, reacts with amine groups of proteins and cross-links adjacent proteins
• Electron microscopy


  Provides much greater magnification and resolution of very small cellular structures, requires careful fixation to preserve additional "ultrastructural" detail
• Preparing tissue for electron microscopy

  1. Glutaraldehyde treatment

  2. Immersion in buffered osmium tetroxide
• Paraffin


  Used routinely for embedding tissues for light microscopy
• Plastic resins


  Used for embedding tissues for both light and electron microscopy
• Embedding and sectioning


  1. Dehydration

  2. Clearing

  3. Infiltration with paraffin or plastic resin

  4. Embedding

  5. Trimming

  6. Sectioning on a microtome
• Microtome


  Instrument used for sectioning paraffin-embedded tissues for light microscopy
• Cryostat


  Microtome used to section rapidly frozen tissue samples for quick analysis
• Staining


  Methods used to make various tissue components conspicuous and distinguishable under the microscope
• Basophilic


  Cell components with a net negative charge that have an affinity for basic dyes
• Acidophilic


  Cationic cell components that stain more readily with acidic dyes
• Basic dyes


  • Toluidine blue, alcian blue, methylene blue
• Acid dyes


  • Eosin, orange G, acid fuchsin
• Hematoxylin and eosin (H&E)


  Simple and commonly used staining method, hematoxylin stains DNA and RNA-rich areas blue/purple, eosin stains other cytoplasmic structures and collagen pink
• Periodic acid-Schiff (PAS) reaction

  Stains carbohydrate-rich tissue structures purple/magenta
• Bright-field microscopy


  Conventional microscopy using ordinary light passing through the stained tissue specimen
• Light microscope


  • Includes condenser, objective lens, and eyepiece lens to focus light and magnify the image
  • Resolving power of approximately 0.2 μm is the maximum for light microscopes
• Virtual microscopy


  Conversion of bright-field microscopic preparations into digital images for computer-based study