Intended use: For In Vitro Diagnostic and Research Use. The Citrate buffer is intended for use in immunohistochemistry for heat-induced epitope retrieval.
Summary and explanation: The Citrate buffer, is designed for use during heat-induced epitope retrieval (HIER) on formalinfixed paraffin-embedded tissue sections prior to antibody application. In immunohistochemistry (IHC), protein cross-links are formed during formalin or other aldehyde fixation. These cross-links mask the antigenic sites in tissue specimens and results in weak or false negative staining in immunohistochemical detection of proteins. The use of this citrate buffer in combination with heat ensures that the antigenicity of proteins modified, thus enhancing staining intensity of antibodies. For the recommended pre-treatment technique, please refer to the individual antibody's instructions for use. This buffer is a 10x concentrate solution and must be diluted 1:10 with deionized water before using. The final readyto-use solution should have a pH of 6.0 ± 0.3. The buffer can be used for a maximum of three runs within five days after dilution. Also available in 250 ml, 500 ml en 1000 ml.
Reagents provided: 100 ml 10x concentrate solution. Citrate buffer, Heat-Induced Epitope Retrieval (10x, Tween20), Red
Storage and handling: Store at room temperature
Principle of method: Heat-Induced Epitope Retrieval
Reagent preparation: Before use dilute 1:10 with deionized water. The final ready-to-use solution should have a pH of 6.0 ± 0,3,
Procedure: 1. Deparaffinize and rehydrate the tissue slides. 2. Fill the PT Module tank with 150 ml of citrate buffer and 1,350 ml of deionized water, to obtain 1,500 ml of ready-to-use solution (1:10 dilution).3. Pre-heat the PT Module until temperature reaches 65°C and place the formalin-fixed slides in slide racks into the PT Module tank.4. Heat the citrate buffer and slides to 98°C and incubate for 20 minutes.5. Cool slides in the PT Module to 65°C.6. Remove slides and cool to room temperature for at least 5 minutes in a PBS or TBS based buffer.7. Continue with staining according to IHC protocol. Note: Alternative heating sources, such as a microwave or a pressure cooker, can be used to replace the PT Module tank. The optimal incubation time for these heating sources should be determined by authorised personnel / researchers.
Expected results: Epitope retrieval
References: Morgan JM,et al (1994) J Pathol. 174(4):301-7/Hoffman, E. A., et al. (2015). Journal of Biological Chemistry, 290(44), 26404–26411.
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