Localization of Transglutaminase Activity

Tissue samples were embedded in optimal cutting temperature (OCT) compound (Scigen Scientific, Gardena, CA, USA) and snap-frozen in liquid nitrogen. Cryosections of 6 µm thickness were cut with a cryostat (Leica CM3050S) and stored at −80 °C. Transglutaminase activity was localized with an in situ assay according to a published protocol [28 (link)], with minor modifications. Briefly, cryosections were incubated with 5 µM Alexa-fluor-555-cadaverine (Thermo Fisher, Waltham, MA, USA), a substrate of transglutaminases, in 0.1 M Tris-HCl pH 7.4 in the presence of 5 mM CaCl₂ for 2 h. In negative control experiments, CaCl₂ was replaced by 5 mM EDTA. The reaction was stopped by incubating the slides in 25 mM EDTA in PBS for 5 min. Nuclei were labeled with 1 µg/mL Hoechst 33258 (Molecular Probes, Eugene, OR, USA). The sections were mounted with Permafluor (Thermo Fisher, Waltham, MA, USA) and examined with an Olympus BX63 microscope. Photographs were taken with an Olympus UC-90 camera.