Immunohistochemistry Techniques


Immunochemistry is the identification of a certain antigen in a histological tissue section or cytological preparation by an antibody specific to that antigen. Immunohistochemistry refers specifically to histological tissue sections.

Immunohistochemistry Techniques

Immunohistochemistry Techniques uses antibodies, reagents and stains for the diagnosis and research of cancer. The common nuclear counterstains are: Hematoxylin, Light Green, Fast Red, Toliudine Blue and Methylene Blue. Also an Alum-Mordant base on Hematoxylin is used such as Harris's Hematoxylin (now is offered without mercury). Mayer's Hematoxylin is one of the most popular mordants used in immunohistochemistry as well as Gill's Hematoxylins that are classified as 1,2,3.

Immunohistochemistry Techniques uses different methods and approaches. The specimen needs to be well fixed. One of the most popular fixatives is 10% Neutral Formalin and Zinc Formalin. A lso in immunohistochemistry, a transport solution is needed to transport the specimen. The most popular is Michel's Immunofluorescence Working.

Immunohistochemical techniques detect antigens in tissue sections by means of immunological and chemical reactions. This technique is highly sensitive and specific and can detect a wide variety of antigens in multiple animal species. This chapter reviews common immunohistochemical methods used in the characterization of normal and pathologic tissue and the reagents used. Pretreatments such as blocking steps for endogenous activities and antigen retrieval are included. Standard procedures on formalin-fixed, paraffin-embedded tissues as well as method standardization for new antibodies and troubleshooting are emphasized.

  • 1. Deparaffinize and rehydrate sections as follows: 2 x 10 min in xylene, 2 min in 100% ethanol, 2 min in 95% ethanol, 2 min in 80% ethanol, 2 min in 70% ethanol, 5 min in tap water and 5 min in distilled water.
  • 2. Block endogenous peroxidases by soaking slides in a solution mixtured with methanol and 30% H2O2 for 10 min at room temperature. Then wash the slides 5 min in distilled water, 5 min in PBS.
  • 3. Heat Induced Epitope Retrieval: Antigen retrieval by microwavl irradiation 2x5min. Then remove the staining dish to room temperature and allow the slides to cool for 20 minutes.
    Note:The AR solution (pH 6.0)is mixture of Sodium Citrate Buffer and Citrate Buffer. Wash the slides 3x5min with PBS.
  • 4. Block in 10% normal goat serum in PBS for 1 hour at 37℃.
  • 5. Incubate sections with primary antibody at appropriate dilution in PBS overnight at 4 °C.
  • 6. Wash the slides 3x5min with PBS.
  • 7. Incubate sections with HRP-conjugated secondary antibody at appropriate dilution in PBS for 30 min at 37℃.
  • 8. Wash the slides 3x3min with PBS.
  • 9. Develop with DAB till the color is appropriate, then rinse the slides under tap water gently for about 1- 2 min.
  • 10. Counterstain in hematoxylin.
  • 11. 2 x2min in 100% ethanol, 2 x5min in xylene, mount sections with neutral balsam.