title: A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection.
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.
authors.affiliation: *Pathology Azienda Ospedaliera San Gerardo †Dipartimento di Chirurgia e Medicina Traslazionale, Universitá degli Studi di Milano-Bicocca, Monza, Italy.
authors.forename: Carla R
authors.initials: CR
authors.lastname: Scalia
authors.forename: Rossella
authors.initials: R
authors.lastname: Gendusa
authors.forename: Giorgio
authors.initials: G
authors.lastname: Cattoretti
mesh_terms: D000941:Antigens / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry
chemical_list: D000941:Antigens