Routine Paraffin Cytoplasmic, Membrane Antigens and Nuclear Antigens

Purpose
Immunohistochemistry is used to identify a long list of antigens. It is a very effective and flexible technique that is dependent upon the specificity and sensitivity of the primary antibody. If one has an antibody that identifies the fixed, processed antigen, any number of secondary antibodies and/or amplification techniques can be used to identify the antigen in paraffin sections.
Principle
The tissue is processed and unstained paraffin sections are mounted on glass slides. Most procedures call for "antigen retrieval" using microwave ovens and citrate buffers. The incubation in methanol and hydrogen peroxide is designed to "kill" the endogenous peroxidases and eliminate background staining. The appropriate dilution of primary antibody is placed in solution on the deparaffinized sections and exposed for an appropriate time interval (depending on the affinity and avidity of the antibody and the preservation and concentration of the antigen). The secondary antibody systems are then placed on the slide. The procedures provided below illustrate the use of the ABC kits but many different protocols, signal amplification systems and indicator systems are available. The new mouse-on-mouse systems permit the use of some mouse monoclonal antibodies on mouse tissue.
Fixative
In general, tissue fixed in paraformaldehyde for less than 48 hours provides the best results. However, different antigens will require different protocols. Post fixation of formalin or paraformaldehyde fixed tissue with B5 or other heavy metal fixative can "retrieve" some antigens.

Paraffin mounted slides

  1. Xylene 5 min.
  2. Xylene 5 min.
  3. Xylene/Lugol’s 4 min. *
  4. Absolute ETOH 3 min.
  5. Absolute ETOH 3 min.
  6. MEOH/H2O2(0.3%) 20 min.
  7. Absolute ETOH 1 min.
  8. 95% ETOH 2 min.
  9. 70% ETOH 2 min.
  10. H2O (running) 2 min.
  11. Dist. H2O 2 min.
  12. PBS (x2) 5 min.
  13. Normal Equine Serum 10% 20 min.<
  14. Primary abs. (Dilutions vary) Overnight (4 ° Celsius)
  15. PBS (x2) 5 min.
  16. Equine Anti-Mouse Biotin Conjugate 60 min.
  17. PBS (x2) 5 min.
  18. ABC - Elite 30 min.
  19. PBS (x2) 5 min.
  20. DAB (Vector-Brown) 3-5 min.
  21. Mayer’s Hematoxylin 1 min.
  22. Tap H2O 5-10 min.
  23. Dehydrate, clear and coverslip
  24. *B5 Fixed Tissue only
  25. NOTE: Tissue sections are placed in a 55 Celsius oven for 30 minutes or overnight.

Immunoperoxidase Staining (Nuclear Antigens)

  1. Xylene 5 min. RoomTemp.
  2. Xylene 5 min. RoomTemp.
  3. ETOH 3 min. RoomTemp.
  4. ETOH 3 min. RoomTemp.
  5. H202 + 3% Methanol 20 min. RoomTemp..
  6. ETOH 2 min. RoomTemp.
  7. ETOH 2 min. RoomTemp..
  8. H2O tap 5 min. RoomTemp..
  9. distilled H2O 5 min. RoomTemp..
  10. Citrate Buffer 4 min. microwave*
  11. Citrate Buffer 4 min. microwave*
  12. Citrate Buffer 4 min. microwave
  13. Cool down 15 min. RoomTemp
  14. PBS 5 min. RoomTemp
  15. PBS 5 min. RoomTemp.
  16. N horse serum 20 min. RoomTemp.
  17. Primary antibody overnight 4 C
  18. PBS 5 min. RoomTemp.
  19. PBS 5 min. RoomTemp.
  20. Secondary (BHAM) 1:800 60 min. RoomTemp.
  21. PBS 5 min. RoomTemp.
  22. PBS 5 min. RoomTemp.
  23. Tertiary ABC 1:50 30 min. RoomTemp.
  24. PBS 5 min. RoomTemp.
  25. PBS 5 min. RoomTemp.
  26. DAB 3-5 min. RoomTemp.
  27. H2O running tap 5 min. RoomTemp.
  28. Hematoxylin-Mayers 30 seconds RoomTemp.
  29. H2O running tap
  30. Dehydrate, clear, coverslip