A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection

Detection by immunohistochemistry of antigens relies on reproducibly optimal preanalytical and analytical variables such as fixation conditions, antigen retrieval (AR), and the resolutive power of the detection system. There is a need to improve immunodetection on routinely fixed and embedded material, particularly for scarcely represented but relevant antigens. We devised a 2-step method and applied it to a panel of antigens of common use for diagnosis, prognosis, individualized therapy use, or research. The first step consists of a 10 minutes. Incubation at 95°C with a modified Laemmli extraction buffer. This was followed by a traditional AR method. Detection of the vast majority of antigens was improved over a simple AR with preservation of tissue integrity, as shown by quantitative image analysis. The mechanism underlying the improved detection may be controlled denaturation followed by heat-mediated retrieval, a method we dubbed "antigen relaxing" and which will improve routine detection of scarce antigens in formalin-fixed, paraffin-embedded material.