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:
- Deparaffinize sections in 2 changes of xylene, 5 minutes each.
- Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each.
- Rinse in distilled water.
- 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).
- Allow sections to cool at room temperature for 10 minutes.
- Rinse sections in PBS Tween 20 for 2x2 min.
- Block sections with for 30 minutes.
- Perform avidin/biotin blocking if necessary.
- Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 ºC.
- Rinse sections with PBS Tween 20 for 2x2 min.
- Block sections with peroxidase blocking solution for 10 minutes.
- Rinse with PBS Tween 20 for 3x2 min.
- 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