An immunofluorescence method for postembedded tissue in the acrylic resin Technovit 9100 New® using fluorescein isothiocyanate secondary detection

Abstract

Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin components and stabilizers are a source of additional strong native fluorescence that overlaps with absorption and emission spectra of commonly used green fluorophores, the unfixed tissue is also subject to native fluorescence. For tissue embedded in resin, we hypothesized that initially removing the resin and subsequently quenching the native fluorescence from the sample could result in specific immunofluorescence signals. The hypothesis was tested on fixed tissue samples embedded in Technovit 9100 New®. Deacrylated and rehydrated semithin sections from a variety of soft tissues were exposed to a quenching solution prior to immunolabeling. Cryostat sections from snap frozen tissue were also stained to assess whether all antigens investigated in fixed tissue were adequately detected. The secondary detection included antibodies conjugated with fluorescein isothiocyanate. The results were evaluated using conventional dark-field and confocal laser scanning microscopy. Both forms of microscopy confirmed the considerable lowering of the native fluorescence associated with the resin and fixed tissue samples with enhanced specific signal. The cryostat tissue sections using the same antibodies in equivalent concentrations con- firmed labeling of the same cellular sites as those observed in the fixed tissue. This article describes a method for immunofluorescence labeling in Technovit 9100 New resin embedded tissue and suggests the likely chromogenic elements generating autofluorescence.