The synergistic relationship of enzyme and HIER – For strong, specific staining of difficult antibodies

The synergistic relationship of enzyme and HIER
For strong, specific staining of difficult antibodies

With the increased use of heat-induced epitope retrieval (HIER) for formalin-fixed paraffin-embedded (FFPE) tissues on automated
immunohistochemistry (IHC) instruments, enzymatic digestion use has decreased. Automated instruments often lack the capacity to exceed
100°C, resulting in less than optimal results compared to offline antigen retrieval methods. The use of enzymes alone for epitope retrieval lacks
in comparison to HIER as higher concentrations and incubation times are needed to produce appropriate expression but run the risk of tissue
over-digestion. Using a synergistic relationship of HIER and enzymatic digestion, automated instruments can make staining equivalent to offline
pressured HIER methods.

Using automated and semi-automated IHC instrumentation, Desmoglein 3, MSH6, and CD4 slides were stained with and without a weak postretrieval enzymatic digestion utilizing Pronase and Trypsin. With the use of Pronase, Desmoglein 3 and CD4 exhibited significant increase in
staining. Trypsin increased the staining of MSH6 by one grade increase. All antibodies using this synergistic relationship not only enhanced
staining intensity but exhibited increased sensitivity when compared to the control with offline HIER using Biocare Medical’s Decloaking
Chamber™ NxGen.

A synergistic relationship between HIER and enzyme digestion was easily achieved without compromising tissue integrity. The addition of
enzymatic digestion after HIER proved to be a superior method that not only increased staining intensity but allowed for the detection of
positive cells that might have otherwise been missed during review. This combination pretreatment approach is ideal for problematic or difficult
antibodies stained on automated IHC platforms with a less than optimal online HIER.

 

The addition of enzymatic digestion after HIER proved to be a
superior method that increased staining intensity. 

 

 Without Enzymatic Digestion                                                                                         With Enzymatic Digestion

                                               

CD4 (RM) in tris-based buffer without post-retrieval enzymatic digestion.               CD4 (RM) in tris-based buffer with post-retrieval enzymatic digestion with high
Low staining intensity and decreased sensitivity can be seen.                                 increase in staining intensity

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