title: Antigen Masking During Fixation and Embedding, Dissected.
abstract: Antigen masking in routinely processed tissue is a poorly understood process caused by multiple factors. We sought to dissect the effect on antigenicity of each step of processing by using frozen sections as proxies of the whole tissue. An equivalent extent of antigen masking occurs across variable fixation times at room temperature. Most antigens benefit from longer fixation times (>24 hr) for optimal detection after antigen retrieval (AR; for example, Ki-67, bcl-2, ER). The transfer to a graded alcohol series results in an enhanced staining effect, reproduced by treating the sections with detergents, possibly because of a better access of the polymeric immunohistochemical detection system to tissue structures. A second round of masking occurs upon entering the clearing agent, mostly at the paraffin embedding step. This may depend on the non-freezable water removal. AR fully reverses the masking due both to the fixation time and the paraffin embedding. AR itself destroys some epitopes which do not survive routine processing. Processed frozen sections are a tool to investigate fixation and processing requirements for antigens in routine specimens.
authors: Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).
authors: Carla Rossana
authors: CR
authors: Scalia
authors: Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).
authors: Giovanna
authors: G
authors: Boi
authors: Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).
authors: Maddalena Maria
authors: MM
authors: Bolognesi
authors: Azienda Socio Sanitaria Territoriale Monza, Monza, Italy (LR, BEL, GC).
authors: Lorella
authors: L
authors: Riva
authors: Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).
authors: Marco
authors: M
authors: Manzoni
authors: Laboratory of Translational Cell and Tissue Research, University of Leuven, KUL, Leuven, Belgium (LD, FMB).
authors: Linde
authors: L
authors: DeSmedt
authors: Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).|Laboratory of Translational Cell and Tissue Research, University of Leuven, KUL, Leuven, Belgium (LD, FMB).
authors: Francesca Maria
authors: FM
authors: Bosisio
authors: Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).
authors: Susanna
authors: S
authors: Ronchi
authors: Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).|Azienda Socio Sanitaria Territoriale Monza, Monza, Italy (LR, BEL, GC).
authors: Biagio Eugenio
authors: BE
authors: Leone
authors: Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).|Azienda Socio Sanitaria Territoriale Monza, Monza, Italy (LR, BEL, GC).
authors: Giorgio
authors: G
authors: Cattoretti
mesh_terms: D000941:Antigens / Q000032:analysis*; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005629:Frozen Sections / Q000379:methods; D006801:Humans; D016612:Paraffin Embedding / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D000941:Antigens; D000939:Epitopes
keywords: FFPE; antigen retrieval; fixation; formalin; immunostaining