Trypsin Antigen Retrieval Protocol




Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The trypsin based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.


Solutions and Reagents:  


Trypsin Solution:


Trypsin Stock Solution (0.5% in Distilled Water):

Trypsin -------------------------------- 50 mg

DDistilled water ------------------------ 10 ml

Mix to dissolve. Store at -20 ºC.


Calcium Chloride Stock Solution (1%):

CCalcium chloride --------------------- 0.1 g

Distilled water ----------------------- 10 ml

Mix well and store at 4 ºC.


Trypsin Working Solution (0.05%):

Trypsin stock solution (0.5%) ------------ 1 ml

Calcium chloride stock solution 1% ------ 1 ml

Distilled Water ----------------------------- 8 ml

Adjust pH to 7.8 with 1N N NaOH. Store at 4 ºC for one month or -20 ºC for long term storage



  1. Deparaffinize sections in 2 changes of xylene, 5 minutes each.

  2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each.

  3. Rinse in distilled water.

  4. Cover sections with trypsin working solution and incubate for 10-20 minutes at 37 °C in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user).

  5. Allow sections to cool at room temperature for 10 minutes.
  6. Rinse sections in PBS Tween 20 for 2x2 min.
  7. Block sections with for 30 minutes.
  8. Perform avidin/biotin blocking if necessary.
  9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
  10. Rinse sections with PBS Tween 20 for 2x2 min.
  11. Block sections with peroxidase blocking solution for 10 minutes.
  12. Rinse with PBS Tween 20 for 3x2 min.
  13. Proceed to standard immunohistochemistry protocol.

Notes: This method tends to produce tissue damage so incubation time is import factor to consider when using this method. Select appropriate incubation time for a specific application.



1. Ashton-Key M, Jessup E, Isaacson PG (1996) Immunoglobulin light chain staining in paraffin-embedded tissue using a heat mediated epitope retrieval method. Histopathology. 29(6):525-31. PubMed Abstract

2. Kashima K, Yokoyama S, Daa T, Nakayama I, Nickerson PA, Noguchi S (1997) Cytoplasmic biotin-like activity interferes with immunohistochemical analysis of thyroid lesions: a comparison of antigen retrieval methods. Mod Pathol. 10(5):515-9. PubMed Abstract

3. Frost AR, Sparks D, Grizzle WE (2000) Methods of antigen recovery vary in their usefulness in unmasking specific antigens in immunohistochemistry. Appl Immunohistochem Mol Morphol. 8(3):236-43. PubMed Abstract