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Hydrochloric Acid Antigen Retrieval Protocol Last update on July 17, 2012 under Immunohistochemistry
Contents
Introduction
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Hydrochloric acid antigen retrieval is used with paraffin embedded, formalin or paraformaldehyde-fixed tissues. When tissues are 1. Introduction immersed in cross-linking agents, such as from paraformaldehyde, these agents will covalently cross-linked proteins, resulting a 2. Materials reduction in the available epitopes for antibody binding. The result is weak or negligible levels of protein detection. Consequently, 3. Procedure the use of a relatively strong hydrochloric acid solution has been shown to greatly improve binding for some antibodies. However, not all antibodies respond equally well to a low pH solution treatment, and several antigen retrieval condition should be tested when optimizing stainng conditions. Materials Hydrochloric acid solution, 2N Prepare the solution by adding 20 ml of 10N (concentrated) HCl to 80 ml of distilled water. Be sure to mix the solution well; the pH should be in the range of 0.5-1.0. Procedure 1. Deparaffinize and rehydrate tissue sections. 2. Incubate sections using the 2N hydrochloric acid solution for 10 to 20 minutes at room temperature. A range of times (suggestion 10, 15, and 20 minutes) incubation time should be tested to determine an optimal antigen retrieval time. 3. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to neutralize acidity. 4. Proceed with the tissue blocking for immunohistochemistry. Share this:
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