principles of staining

• Staining


  Process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cells
• Affinity of staining


  • Acidic structures: Basic dyes
  • Basic structures: Acidic dyes
• Methods of Staining


  • Direct staining
  • Indirect staining
  • Progressive staining
  • Regressive staining
• Direct staining


  Process of giving color to the sections using aqueous / alcoholic dye solutions
• Indirect staining


  Action of dye is intensified by adding another agent or a mordant
• Mordant


  Serves as a link or bridge between tissue and the dye to make staining reaction possible
• Accentuator


  Accelerates or hastens the speed of staining reaction by increasing staining power and selectivity. It is not essential to the chemical union of tissue and the dye
• Progressive staining


  1. Tissue elements are stained in a definite sequence

  2. Staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained

  3. Less favored than regressive staining
• Regressive staining


  1. The tissue is first overstained to obliterate the cellular details

  2. Excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained
• Differentiation / Decolorization


  Selective removal of excess stain from the tissue during regressive staining
• Orthochromatic staining


  Tissues are stained in color shades that are similar to the color of the dye itself
• Metachromatic staining


  Staining with a color that is different from that of the stain itself (metachromasia)
• Blueing


  Alum hematoxylin stains nuclei with red color, which is converted to the familiar blue black when the section is washed in a weak alkali solution
• Metallic Impregnation


  Process where a specific tissue element is demonstrated by colorless solutions of metallic salts which are reduced by the tissues producing an opaque, usually, black deposit on the surface of the tissue
• Negative Staining


  Stains are not taken up by their tissue targets. The shapes and structures are disclosed by outlining or filling them with a stain
• Vital Staining


  Selective staining of living cell constituents
• Intravital Staining


  Done by injecting the dye into any part of the animal body
• Staining methods for Frozen sections


  • Hematoxylin-Eosin method
  • Thionine method
  • Polychrome Methylene Blue method
  • Alcoholic Pinacyanol method
• H&E staining of Frozen Sections (Progressive Staining)

  1. Harris hematoxylin

  2. Blue in ammonia water

  3. Counterstain with 5% aqueous eosin or 1% alcohol eosin

  4. Dehydrate in increasing concentrations of alcohol

  5. Clear with xylene
• Collodionization of Sections


  Coating the slide with dilute (thin) celloidin solution to form ribbons
• Staining of Celloidin Sections


  Sections treated with 95% alcohol are transferred to a mixture of equal parts of chloroform, absolute alcohol and xylene and mounted in Xam
• Restaining faded slide


  Subject to a differentiating agent such as 1-2% alcohol
• Restaining of Old Sections


  1. Immerse in xylene for 24 hours or gently heat until the mounting medium begins to bubble

  2. Remove cover slip

  3. Place in xylene for 30 minutes to remove the remaining canada balsam and then bring to water

  4. Immerse in 0.5% potassium permanganate solution for 5 to 10 minutes, rinse in tap water and subsequently immerse in 5% oxalic acid for 5 minutes or until section is decolorized

  5. Wash in running tap water for 5 minutes, then re-stain with the appropriate staining technique