Anti avidin fitc

For hybridization procedure sections were deparaffinized and hydrated in a series of alcohol. Subsequently digestion with pepsin and tissue permeabilization with sodium thiocyanate at 80°C were performed. Then sections were equilibrated in 50% formamide/2xSSC, prehybridized in 45°C, denatured in 80°C and hybridized for 40 h. In brief, post-hybridization procedures were done by incubation sections in 2xSSC and 0.1xSSC in 60°C 2x10 min. Detection of biotin-conjugated probes was performed by the incubation with avidin-Alexa488 (Molecular Probes, Eugene, OR, USA) and anti-avidin-FITC (Sigma Aldrich, St. Louis, MO, USA) antibodies. Detection of digoxygenin- conjugated probes was performed with the anti-digoxigenin – rhodamine sheep antibody (Roche Applied Science, Penzberg, Germany). In order to visualize the nuclei, sections were stained with Hoechst reagent (Molecular Probes). Specimens were examined under Nikon Eclipse 80i fluorescent microscope (Nikon, Tokyo, Japan). To assess the gene status in each section, 200 cells were counted at 600x magnification.