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 Tris-EDTA 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:
Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0):
Tris Base -------------------------------- 1.21 g
EDTA ------------------------------------- 0.37 g
Distilled water -------------------------- 1000 ml (100 ml to make 10x, 50 ml to make 20x)
Mix to dissolve. pH is usually at 9.0 and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.
Note: This buffer
works excellent for many antibodies, but it often gives high
background staining (maybe due to endogenous biotin revealed after
this pretreatment). So primary antibody can often be highly diluted.
It is very useful for low affinity antibodies or when tissue
antigens are not intense.
Procedure:
Deparaffinize sections
in 2 changes of xylene, 5 minutes each.
Note: Microwave or pressure cooker can be used as alternative heating source to replace steamer or water bath.
References: