IHC

• Immunohistochemistry


  Make use of antigen-antibody reactions by directly labeling of the antibody or by means of a secondary labeling method
• Purpose of IHC

  • Detects antigen
  • Detector: Antibodies
  • Most common form of antibody used in IHC: IgG antibodies
• Types of Antibodies used in IHC

  • Polyclonal
  • Monoclonal
• Polyclonal antibodies


  • Produced by different cells
  • Can react with various epitopes
  • May be obtained from laboratory animal sources
  • Main source: Rabbits — immunized to produce antibodies
• Monoclonal antibodies


  • More specific because it is made from individual cells
  • Produces only 1 type of antibody = reacts with only 1 specific type of epitope
  • Animal source: Mice
• Labeling of Antibodies

  • Use of Enzymes
  • Fluorochrome Label
  • Other Labels
• HRP - Horse Raddish Peroxidase


  • Most widely used
  • Alternative: ALP
  • Use of Chromogen: DAB - Diaminobenzidine (resulting color: brown)
  • Use of Chromogen: AEC - 3-amino-9-ethylcarbazole (resulting color: brick red)
• Optimum Incubation Time to link antibody with enzyme peroxidase

  30-60 mins at RT
• FITC - Fluorochrome isothiocyanate


  Needs the use of Fluorescence Microscope
• Lectins


  • Plant or animal proteins which can bind to tissue carbohydrate
  • Can also be used to detect antigens, can also be labelled like antibodies
• Specimens for IHC

  • Cryostat Frozen Sections
  • Processed Specimens
• Cryostat Frozen Sections

  Fixed in a few seconds using ABSOLUTE METHANOL or ACETONE
• Purpose of Fixation

  • Prevent destruction of labile antigenic sites
  • Preserve position of antigens
• Processed Specimens

  • Formalin-fixed spx
  • Paraffin-embedded spx
• Methods of Antigen Retrieval from Processed Tissues

  • Proteolytic enzyme retrieval (PIER)
  • Microwave antigen retrieval / Heat Induced Epitope retrieval
  • Pressure cooking antigen retrieval
  • Autoclave heating
  • Waterbath heating (90degC or 95-98 degC
  • Steamer heating
  • Decloaker heating
  • Combination of microwave and enzyme digestion
• Controls


  • Positive Control
  • Negative Control
  • Internal Tissue Control
• Positive Control


  A tissue section with the antigen being detected
• Negative Control


  To prepare, we omit the primary antibody from the staining schedule
• Internal Tissue Control


  Contains the target antigen not only in the tissue elements but also in the adjacent normal tissue elements
• Epithelial Tumor Markers


  • Keratin
  • Epithelial Membrane Antigen (EMA)
  • Carcinoembryonic Membrane Antigen (CEA)
  • Thyroid Transcription Factor-1 (TTF-1)
  • Prostate Specific Antigen (PSA)
• Keratin


  • Highly sensitive marker for epithelial cells (Epithelial tumors)
  • Cytokeratin 7 (CK7) - Carcinomas of the lung, breast, uterus and ovaries (Serous tumors)
  • Cytokeratin 20 (CK20) - Carcinomas of colon and stomach
  • CK7 and CK20 - Both positive in Transitional carcinomas of the bladder and mucinous ovarian tumor
• Epithelial Membrane Antigen (EMA)


  Determining site tumor - Breast, lung, and kidney adenocarcinomas
• Carcinoembryonic Membrane Antigen (CEA)


  Differentiating adenocarcinoma and mesothelioma - GIT, pancreas, lung, breast, ovary, uterus, and cervix
• Thyroid Transcription Factor-1 (TTF-1)

  Distinguishing lung adenocarcinoma from mesotheliomas
• Prostate Specific Antigen (PSA)


  Prostatic carcinoma, pancreatic and salivary gland tumor
• Intermediate Filament Markers

  • Actin
  • Vimentin
  • Desmin
  • Glial fibrilliary acidic protein (GFAP)
  • Neurofilament (NF)
  • S100 Protein
• Actin


  Muscle differentiation - Smooth, skeletal, and cardiac muscle
• Vimentin


  Melanomas and Schwannomas
• Desmin


  Myogenic tumors (Leiomyoma and Rhabdomyosarcoma)
• Neurofilament (NF)


  Neuronal or neuroendocrine differentiation
• S100 Protein


  Calcium-binding protein that is expressed in: CNS glial cells, Shwann cells, Melanocytes, Chondrocytes, Skeletal and cardiac muscles, Myoepithelial cells
• Neuroendocrine Markers


  • Neuron-specific enolase (NSE)
  • Chromogranin
  • Synaptophysin
• Neuron-specific enolase (NSE)


  Neural or neuroendocrine differentiation
• Chromogranin


  Neuroendocrine differentiation
• Synaptophysin


  • Associated with presynaptic vesicles of neurons
  • Identified in normal neurons and neuroendocrine cells
• Mesenchymal Tumor Markers


  • Muscle-specific actin and myosin; Myo-D1, Myoglobin, and Myogenin
  • CD68 or FAM 56; alpha-1-antitrypsin and alpha-1-antichymotrypsin
  • Endothelia Markers: Factor VII-related antigen, CD31, Ulex Europaeus (UEA)
  • S100 protein Melanosome (HMB-45), Melan-A (MART-1)
  • Best: Leukocyte Common Antigen (LCA) or CD45
• Muscle-specific actin and myosin; Myo-D1, Myoglobin, and Myogenin


  Myogenic tumors (Skeletal muscle)
• CD68 or FAM 56; alpha-1-antitrypsin and alpha-1-antichymotrypsin

  Fibrohistiolytic tumors
• Endothelia Markers: Factor VII-related antigen, CD31, Ulex Europaeus (UEA)


  Vascular tumors (Angiosarcoma)