title: Immunofluorescence and Immunohistochemical Detection of Keratins.
abstract: Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin intermediate filaments are obligatory heteropolymers, which may result in dissociation between RNA and protein expression levels in the event that the homeostasis of the expression of the proper keratin partners is disturbed. Furthermore, variable accessibility of epitopes on keratin polypeptides due to conformational changes may lead to false negative results. Preanalytical effects, such as warm/cold ischemia, fixation, tissue processing, and embedding may result in false negative or inappropriate reactions. An experimental design for how to systematically test preanalytical effects and to validate immunohistochemistry protocols is presented. This kind of evaluation should be performed for each antigen and antibody since the various epitopes recognized by antibodies may behave differently. In this context, one has to be aware that different cell structures may be affected or modified differently by various preanalytical procedures and may thus require different preanalytical and staining protocols.
authors.affiliation: Institute of Pathology, Medical University of Graz, Graz, Austria.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Stumptner
authors.affiliation: Institute of Pathology, Medical University of Graz, Graz, Austria.
authors.forename: Margit
authors.initials: M
authors.lastname: Gogg-Kamerer
authors.affiliation: Institute of Pathology, Medical University of Graz, Graz, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Viertler
authors.affiliation: Institute of Pathology, Medical University of Graz, Graz, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Denk
authors.affiliation: Institute of Pathology, Medical University of Graz, Graz, Austria. Electronic address: kurt.zatloukal@medunigraz.at.
authors.forename: Kurt
authors.initials: K
authors.lastname: Zatloukal
mesh_terms: D000818:Animals; D057075:Enzyme Assays / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis / Q000378:metabolism*
chemical_list: D007633:Keratins
keywords: Double-label indirect immunofluorescence; Epitope masking; Epitope/antigen retrieval; Formalin-fixed paraffin-embedded tissue; Immunohistochemistry; PAXgene; Preanalytic; Standardization