Intended use: Trypsin Pretreatment Kit consists of 2 reagents for the preparation of a trypsin solution used for enzymatic epitope retrieval on formalin-fixed tissue sections on slides. This procedure (sometimes called PIER, Protease Induced Epitope Retrieval) is primarily used in immunohistochemical staining procedures. Trypsin Pretreatment Kit is for research use only.
Summary and explanation: Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Enzymatic digestion with proteolytic enzymes (PIER) restores structures of the epitopes making them more accessible to specific antibodies. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Reagents provided: 30 ml Trypsin Solution 125 ml Trypsin Buffer
Storage and handling: The solutions should be stored at 2-8°C. Please sto re the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by this reagent, please contact our technical support.
Principle of method: The components of this Trypsin Pretreatment Kit allow for the preparation of a buffered trypsin solution for enzymatic epitope retrieval.
Reagent preparation: Mix 1 part Trypsin Solution with 3 parts Trypsin Buffer. • The activity of the resulting trypsin solution can be adjusted by variation of the mixing ratio. Mix the two components in the ratio 1:1 when strong epitope retrieval is desired. • The working solution is stable for at least one week if stored at 2-8°C.
Procedure: Trypsin Pretreatment Kit is suitable for enzymatic epitope retrieval carried out after the dewaxing and rehydration of the sections. 1. Cover deparaffinised and rehydrated tissue sections with trypsin working solution.2. Incubate for 10 - 20 minutes at 37°C. 3. Rinse carefully (3 x) with wash buffer. 4. Proceed with immunohistological staining as usual.
Expected results: During the reaction of the substrate with horse radish peroxidase or alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
References: Ashton-Key M, Jessup E, Isaacson PG Histopathol 29(6):525-31, 1996/Cattoretti G, Pileri S, Parravicini C et al. J Pathol 171(2):83-98, 1993/Nadji M and Morales AR Ann N.Y. Acad Sci 420:134-139, 1983
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