Remove buffer and apply ready-to-use Carezyme-II solution for 5 minutes at 37°C.
Rinse slides in distilled water for 1 minute to stop digestion.
Pronase
Pronase is purchased concentrated from DakoCytomation(http://www.dako.com/). To prepare, aliquot pronase in 100μl volumes and store at -20°C.
Dilute 100µl stock with
9ml of 1X Automation Buffer.
Apply the 0.05% pronase solution for 5 minutes at room temperature.
Rinse in distilled water for 1 minute to stop digestion.
(For Vimentin staining on mouse tissue, the pronase treatment period is 30 minutes.)
Trypsin
Traditionally, enzyme retrieval with trypsin works at a concentration of 0.01% and is conducted at 37°C. The incubation time can vary and is dependent on the tissue type and the sensitivity of the antibody. For development, the tissue is tested at 5-minute intervals, so multiple sections (slides) are required. One important note, trypsin looses 75% of its reactivity within 30 minutes of preparation. If trypsin is the method of choice for retrieval, the concentration and incubation time will be listed for the respective protocol.