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
The HCl 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
Solutions and Reagents:
Hydrochloric Acid (HCl) Solution (2N in Distilled Water, pH 0.6-0.9):
10N (concentrated) HCl ----------------- 20 ml
Distilled water ---------------------------- 80 ml
Mix well and pH should be around 0.6-0.9
Incubate sections for 10-20 minutes at room temperature.
Deparaffinize sections in 2 changes of xylene, 5 minutes each.
Rinse sections in PBS Tween 20 for 2x2 min.
Block sections with for 30 minutes.
Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
Rinse sections with PBS Tween 20 for 2x2 min.
Block sections with peroxidase blocking solution for 10 minutes.
Proceed to standard immunohistochemistry protocol.
Note: This method works well with BrdU and beta amyloid antibody staining.