A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection. Issue 6 (July 2016)

• Record Type: Journal Article
• Authors: Scalia, Carla R.; Gendusa, Rossella; Cattoretti, Giorgio
• Is Part Of: Applied immunohistochemistry & molecular morphology. Volume 24:Issue 6(2016)
• Publication Date: 2016-07
• Subjects: antigen retrieval -- immunohistochemistry -- extraction buffer -- denaturing buffer -- FFPE; Diagnostic immunohistochemistry -- Periodicals; Immunohistochemistry -- Periodicals; Cells -- Morphology -- Periodicals; Molecular diagnosis -- Periodicals; 616.079
• DOI: 10.1097/PAI.0000000000000203
• Languages: English
• ISSNs: 1541-2016

Abstract

Abstract : 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. Abstract : Supplemental Digital Content is available in the text.