Proteinase K 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 proteinase K 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:

 

Proteinase K Solution (Method 1) (20 ug/ml in TE Buffer, pH 8.0):

 

      TE Buffer (50mM Tris Base, 1mM EDTA, 0.5% Triton X-100, pH 8.0):

                   Tris Base -------------------------------- 6.10 g

          EDTA ------------------------------------- 0.37 g

          Triton X-100 ---------------------------- 5 ml

          Distilled water ------------------------- 1000 ml

          Mix to dissolve. Adjust pH 8.0 using concentrated HCl (10N HCl). Store at room temperature.

 

      Proteinase K Stock Solution (20x, 400 ug/ml or 12 units/ml):

          Proteinase K (30 units/mg)----------- 0.008 g (8 mg)

          TE Buffer, pH8.0 ---------------------- 10 ml

          Glycerol --------------------------------- 10 ml

          Add proteinase K to TE buffer until dissolved. Then add glycerol and mix well. Aliquot and store at –20 ºC for 2-3 years.

 

      Working Solution (1x, 20 ug/ml or 0.6 units/ml):

          Proteinase K Stock Solution (20x) ------ 1 ml

          TE Buffer, pH8.0 ------------------------- 19 ml

          Mix well. This solution is stable for 6 month at 4 ºC.

 

Proteinase K Solution (Method 2) (20 ug/ml in TE-CaCl2 Buffer, pH 8.0):

 

      TE-CaCl2 Buffer (50mM Tris Base, 1mM EDTA, 5mM CaCl2, 0.5% Triton X-100, pH 8.0):

          Tris Base -------------------------------- 6.10 g

          EDTA ------------------------------------ 0.37 g

          CaCl2 ------------------------------------ 0.56 g

          Triton X-100 ---------------------------- 5 ml

          Distilled water ------------------------- 1000 ml

          Mix to dissolve. Adjust to pH 8.0 using concentrated HCl (10N HCl). Store this buffer at room temperature.

 

      Proteinase K Stock Solution (20x, 400 ug/ml or 12 units/ml):

          Proteinase K (30 units/mg) ----------- 0.008 g (8 mg)

          TE-CaCl2 Buffer, pH8.0 --------------- 10 ml

          Glycerol --------------------------------- 10 ml

          Add proteinase K to TE-CaCl2 buffer until dissolved. Then add glycerol and mix well. Aliquot and store at –20 ºC for 2-3 years.

 

      Working Solution (1x, 20 ug/ml or 0.6 units/ml):

          Proteinase K Stock Solution (20x) ------ 1 ml

          TE-CaCl2 Buffer, pH8.0 ------------------ 19 ml

          Mix well. This solution is stable for 6 month at 4 ºC.

 

Procedure:

  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 Proteinase K working solution and incubate 10-20 minutes at 37 ºC 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 ºC.
  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:

1. This method tends to cause tissue damage for under-fixed tissues. Select appropriate incubation time (5-20 minutes) and temperature (20 ºC to 60 ºC) for a specific application and do not over-digest tissues especially for the under-fixed tissues.

2. Method 2 contains CaCl2, where the Ca2+ can activate proteinase K enzyme by 20-25%, therefore increase enzyme activity.

 

References:

 

1. Ramos-Vara JA, Beissenherz ME. Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers. J Vet Diagn Invest. 2000 Jul;12(4):307-11. PubMed Abstract

2. Proteinase K - Enzyme Explorer (Sigma)

3. Proteinase K Wikipedia Entry