“…Amniotic membranes were embedded in Tissue-Tek (Sakura, Tokyo, Japan), snapfrozen in isopentane cooled in liquid nitrogen vapor and stored at −70 • C. Five µm acetone-fixed cryosections were cut on a cryostat CM1860 UV (Leica Microsystems, Wetzlar, Germany), put on SuperFrost/Plus slides (Thermo Fisher Scientific, Darmstadt, Germany), and were kept at −40 • C until labeling. Later, the sections were processed as described elsewhere [33]. Briefly, they were blocked with 10% normal rabbit serum (Life Technologies, Carlsbad, CA, USA) for 1 h at RT, incubated with anti-HMGB1 rabbit polyclonal antibodies (Novus Biologicals, Centennial, CO, USA) for 16 h at 4 • C, and labeled with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies (Life Technologies) for 2 h at RT After embedding in ProLong Gold Antifade Reagent (Life Technologies), the sections were evaluated under an Olympus BX 40 microscope with an Olympus Camedia C-2000 digital camera (Olympus, Tokyo, Japan).…”