Immunocytochemistry

• Immunochemical technique


  Routinely used for the identification of specific or highly selective cellular epitopes or antigens in frozen or paraffin-embedded tissues

  
• Immunocytochemistry


  The use of labeled antibodies to directly detect tumor markers in tissue

  
• Immunocytochemistry


  Used to detect organisms in cytologic preparations such as fluids, sputum samples, and fine needle aspirates
• Antibodies


  Serum factors in the blood formed in response to foreign substance exposure, also known as immunoglobulins

  
• Antibody conjugates


  Antibody that is attached to toxins or radioisotopes to help specifically destroy cancer cells
• Antibody-dependent cell cytotoxicity


  The process of destroying antibody-coated target cells by natural killer cells, monocytes, macrophages, and neutrophils, all of which have specific receptors for antibody

  
• Antigenic variation


  Result of the process of antigen switching
• Antigens


  Macromolecules that are capable of eliciting formation of immunoglobulins (antibodies) or sensitized cells in an immunocompetent host

  
• Epitope


  The key portion of the immunogen against which the immune response is directed; also known as the determinant site

  
• Light Chain


  Small chain in an immunoglobulin molecule that is bound to the larger chain by disulfide bonds. The two types of light chains are called kappa and lambda
• Heavy Chain


  One of the polypeptide units that makes up an immunoglobulin molecule. Each immunoglobulin monomer consists of two heavy chains paired with two light chains
• In situ


  Within the tissue itself
• In situ hybridization


  Binding of a nucleic acid probe to target DNA located in intact cells

  
• Immunohistochemistry (IHC)


  Combines anatomical, immunological and biochemical techniques to identify discrete tissue components by the interaction of target antigens with specific antibodies tagged with a visible label

  
• Immunohistochemistry (IHC)


  • Makes it possible to visualize the distribution and localization of specific cellular components within cells and in the proper tissue context

  
• Steps in IHC methodology


  • Sample preparation
  • Labeling
• Uses of IHC


  • Disease diagnosis
  • Drug development
  • Biological research
• Immunofluorescence


  Often, but not always performed on frozen tissue due to the high background auto-fluorescence seen in formalin fixed paraffin embedded tissue
• Polyclonal Antibodies


  Produced by immunizing an animal with a purified specific molecule (immunogen) that contains the antigen of interest, and collecting immunoglobulin-rich serum after the animal has produced humoral antibody against the antigen

  
• Animals used to produce polyclonal antibodies


  • Rabbit
  • Goat
  • Pig
  • Sheep
  • Horse
  • Guinea pig and others
• Monoclonal Antibodies


  The products of an individual clone of plasma cells

  
• Monoclonal Antibodies


  • Do not cross-react with other molecules
• Preparing tissue for immunohistochemistry

  In certain instances, the tissue must be prepared as a cryostat section and fixed for a few seconds in absolute methanol or acetone, to preserve immunological activity and prevent destruction of some of the labile antigenic sites
• Methods to retrieve masked antigens in routinely processed tissue

  • Proteolytic enzyme digestion
  • Microwave antigen retrieval
  • Microwave and trypsin antigen retrieval
  • Pressure cooker antigen retrieval
• Proteolytic Enzyme Digestion


  1. Formalin fixed paraffin sections are pre-treated with proteolytic enzymes to break down formalin cross-linking and unmask and allow certain antigenic sites to be exposed

  2. The most common enzymes used are trypsin and protease
• Paraffin Sections


  1. Deparaffinize sections in xylene

  2. Hydrate with 100% ethanol

  3. Hydrate with 95% ethanol

  4. Rinse in distilled water

  5. Follow procedure for pretreatment as required
• Pre-treatment of Tissue Sections


  1. Rinse sections in PBS-Tween

  2. Serum Blocking: Incubate sections with normal serum block

  3. Primary Antibody: Incubate sections with primary antibody

  4. Peroxidase Blocking: Incubate sections in peroxidase blocking solution

  5. Secondary Antibody: Incubate sections with biotinylated secondary antibody

  6. Detection: Incubate sections in streptavidin-HRP

  7. Chromogen/Substrate: Incubate sections in DAB solution

  8. Counterstain if desired

  9. Dehydrate and clear
• Immunohistochemistry procedure


  1. Incubate in dilution buffer for 1 hour at room temperature or overnight at 4 °C

  2. Rinse in PBS-Tween 20

  3. Incubate in peroxidase blocking solution for 10 minutes at room temperature

  4. Rinse in PBS-Tween 20

  5. Incubate with biotinylated secondary antibody in PBS for 30 minutes at room temperature

  6. Rinse in PBS-Tween 20 3 times for 2 minutes each

  7. Incubate in streptavidin-HRP in PBS for 30 minutes at room temperature

  8. Rinse in TBS 3 times for 2 minutes each

  9. Incubate in DAB solution for 1-3 minutes

  10. Rinse in PBS-Tween 20 2 times for 2 minutes each

  11. Counterstain if desired

  12. Rinse in distilled water

  13. Dehydrate through 95% ethanol and 100% ethanol

  14. Clear in xylene

  15. Coverslip with mounting medium
• Heat-induced epitope retrieval (HIER)


  A pretreatment method used to improve staining results by recovering antigen reactivity in formalin fixed paraffin embedded tissue
• HIER process


  1. Thermal energy breaks crosslinks that bind surrounding proteins or peptides to the antigen, unmasking the epitope

  2. Thermal energy removes bound calcium ions from sites of cross-links
• HIER heating sources


  • Microwave
  • Vegetable steamer
  • Pressure cooker
  • Water bath
• The higher the temperature of the HIER solutions, the more effective the recovery of the epitope
• Microwave antigen retrieval


  Boiling of formalin-fixed deparaffinized sections in solutions like citrate buffer, EDTA or Tris EDTA
• Microwave antigen retrieval can retrieve antigens previously thought to be lost or destroyed by routine histological processing
• Optimal length of exposure to heat for microwave antigen retrieval is 20 minutes for most antigens and fixation protocols
• Boiling of poorly fixed material often damages nuclear details, and fibrous and fatty tissues tend to detach from the slide
• Amplification of nucleic acids from paraffin-embedded material by PCR is used to detect viral genomes and oncogene mutations

  
• Choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification
• Pressure cooking antigen retrieval


  An alternative to microwave antigen retrieval that is less time consuming and allows for more consistent recovery of many antigens
• Uses of primary antibodies against numerous antigens


  • Diagnosis of tumors
  • Determination of tumor type
  • Evaluation of proliferation potential
  • Identification of infectious agents
  • Prognostic and therapeutic implications
  • Other aspects of diagnostic pathology