Proteolytic Enzyme Digestion

Enzyme Digestion and Relatively Low Temperature (80°C)-Assisted Antigen Retrieval 

Surgical breast biopsy specimens are first fixed with neutral buffered formalin (4%) for 4-6 hr, followed by zinc-formalin for 2 hr. Paraffin sections (5 xm thick) are placed on silane-coated slides, dried on a slide warmer (60°C) for 1 hr and then in an oven (60°C) for an additional 1 hr. Deparaffinized sections are digested with 0.1% trypsin in PBS at 37°C for 15 min. The sections are placed in 10 mM citrate buffer (pH 6.0) and transferred into a water bath (80° or 90°C) for 2 hr. After a 20-min cooling period, the sections are rinsed 

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It is difficult to use proteolytic enzyme digestion with this treatment, but if needed, the sections should be digested following the microwave heating because boiling enzymatically digested tissue results in tissue loss. A milder form of this method is to boil the citric acid buffer only and incubate the sections in the preheated solution, or steam, for 15 min, or sections can be heated to 80°C in this buffer and kept overnight with similar results (11). 

The combination of proteolytic enzyme digestion and heat-based treatments has been reported however, this increases the susceptibility of the tissues for disintegration. Significant shortening of the digestion time and reduction of the enzyme concentration should be undertaken when testing such protocols. Furthermore, reproducible proteolytic enzyme treatments require optimal conditions for individual tissues, which make these protocols difficult to perform. 

Changes in soluble proteins of peanut flour caused by proteolytic enzyme digestion as detected by gel electrophoresis... 

Demasking protocol is inappropriate or has been omitted. Some tissue antigens require proteolytic enzyme digestion or heat induced antigen retrieval performed prior to staining. The need for pretreatment depends on the type and extent of fixation, specific characteristics of the antigen and the type of antibody used. Use the pretreatment method recommended by the manufacturer. No single pretreatment is suitable for all applications. 29-33, 51-65... 

Proteolytic enzymes digested, intrinsic factor-related binder... 

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