STAINING

• Staining


  The process of applying dyes on the section to see and study the architectural pattern of the tissue and physical characteristics of cells
• Most cells are colorless and transparent therefore, histological sections have to be stained to make cells visible
• Dye affinity


  • Acidic constituents have greater affinity with basic dyes (e.g. nucleus)
  • Basic constituents take up more of the acidic dyes (e.g. cytoplasm)
• Many dyes require a mordant, which is a chemical compound that reacts with the stain to form an insoluble, colored precipitate on the tissue and make staining reaction possible
• The colors of the stains are NOT the real color of a particular tissue

  
• Hematoxylin and Eosin (H&E)

  • Histologic stain used by majority of routine histology because it is quick, cheap, and informative
  • Hematoxylin stains nuclear details
  • Eosin stains cytoplasmic details and tissue architecture
• Staining of paraffin sections

  1. Deparaffinization

  2. Removing xylene

  3. Replacing alcohol with water before staining
• After staining, the section is again dehydrated with increasing grades of alcohol and cleared with 2 changes of xylene to prepare for mounting
• Prolonged exposure in alcohol can remove stains, so sections should not be allowed to stay in alcohol for a very long time
• Sections may float off the slide during staining if slides are dirty or greasy, or if sections have not been left in the paraffin oven long enough to dry and be fixed on the slide
• Staining categories

  • Histological
  • Histochemical/Histochemistry
  • Immunohistochemical
• Histological staining


  • Tissue components are demonstrated by direct interaction with a dye or staining solution
  • Active tissue component is colored
• Histochemical/Histochemistry staining


  • Tissue components are studied through chemical reaction
  • Demonstrates chemical components (e.g. fats, carbohydrates)
  • Permits localization of specific tissue substance
• Immunohistochemical staining


  • A combination of histochemical and immunologic techniques
  • Uses antibodies to detect and demonstrate antigens and phenotypic markers
  • Used for diagnosis of abnormal cells, localization of biomarkers, and detection of specific molecular markers
• Crucial factors that can affect the results of immunohistochemical staining protocols include degree of autolysis, fixation medium, fixation duration, incubation period, and concentration of selected antibodies
• Recommended fixative for immunohistochemistry is maximum of 4% neutral buffered formaldehyde solution, with a fixation time up to a maximum of 48 hours
• Microwave-based fixation of tissue in formaldehyde may have adverse effect on immunohistochemical staining
• Direct staining


  Also known as "simple staining", uses 1 aqueous or alcoholic dye solution to produce a color
• Indirect staining


  Tissue components are demonstrated using a dye with the help of either a mordant or an accentuator
• Mordant


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


  Substance that heightens the color intensity and selectivity of the dye, increases the staining power
• Progressive staining


  Involves gradual application of dyes in tissues, with no excess dye and no washing nor decolorization
• Regressive staining


  Tissue is first overstained to obliterate the cellular details, then excess dye is removed by decolorization
• Decolorization (differentiation)


  Refers to the selective removal of excess dye, most commonly using acid alcohol
• Counterstaining (differential staining)


  Involves application of a different dye (color) to produce contrast and background, differentiates between various microorganisms or structures and cellular components
• Regressive staining

  Overstains nuclei, followed by removal of superfluous and excessive color of tissue constituent by acid differentiation
• Decolorization (Differentiation)


  Refers to the selective removal of excess dye
• Decolorizers

  • Acid Alcohol
• Removing acid dye

  Use basic solution
• Removing basic dye


  Use acid solution
• Counterstaining (Differential Staining)


  Involves application of a different dye (color) to produce contrast and background
• Counterstaining uses more than 1 chemical stain
• Counterstaining


  Differentiates between various microorganisms, or structures and cellular components of a single organism
• Counterstaining imparts a distinctive color only to a certain types of bacteria, unlike simple staining which imparts the same color to ALL bacteria with slight variation in color
• Counterstaining


  Usually done by washing the section in simple solution (e.g. water or alcohol), or by use of acids and oxidizing agents
• Removing acid dye

  Use alkaline medium
• Alcohol acts as a differentiator for both basic and acidic dye
• Applications of Differential Staining

  • WBC Differential
  • Gram Stain
• Gram Stain

  • Crystal Violet, and Fuchsin or Safranin
  • Gram (+) - Violet (due to the large peptidoglycan on the outer surface of cell)
  • Gram (-) - Red
• Eosin


  Acidic dye for cytoplasm (basic component)