Proteolytic Enzyme Digestion

The protein cross links formed during formalin fixation can be partially disrupted by the use of proteolytic enzymes of which trypsin is the most widely used. Trypsinisation time is extremely important and is proportional to the specimen fixation time. There is a very fine balance between over and under digestion. Trypsin is optimally active at 37 o C and at pH7.8. The reaction rate is improved by the addition of the co-enzyme calcium chloride (0.1%). Trypsin only remains active for about 30 minutes, therefore if the incubation time exceeds this, the working solution must be replaced. Not all antigens require proteolytic digestion. Furthermore, care must be taken to avoid creating "false" antigenic sites, as some antigens may be altered or destroyed by trypsinisation. In some instances immunostaining may be impaired or completely removed following trypsinisation. Proteolytic digestion has largely been replaced by heat mediated antigen retrieval methods.