Dig rna labeling mix 10 conc

Embryos were dechorionated and fixed in fixation buffer (0.5x PBS, 25 mM EGTA, 4% formaldehyde and 50% Heptane) for 20 min at room temperature. Antisense RNA probes labeled with digoxigenin (DIG RNA Labeling Mix 10 × conc, Roche) and biotin (Biotin RNA Labeling Mix 10 × conc, Roche) were used to detect lacZ and snail RNAs, respectively. Hybridization was performed at 55 °C overnight in hybridization buffer (50% formamide, 5x SSC, 50 μg/ml Heparin, 100 μg/ml salmon sperm DNA, 0.1% Tween-20). Subsequently, embryos were washed with hybridization buffer at 55 °C and incubated with Western Blocking Buffer (Roche) at room temperature for one hour. Then, embryos were incubated with sheep anti-digoxigenin (Roche) and mouse anti-biotin primary antibodies (Invitrogen) at 4 °C for overnight, followed by incubation with Alexa Fluor 488 donkey anti-sheep (Invitrogen) and Alexa Flour 555 goat anti-mouse (Invitrogen) fluorescent secondary antibodies at room temperature for two hours. DNA was stained with Hoechst 33342 (Thermo Fisher Scientific), and embryos were mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Imaging was performed on a Zeiss LSM 880 confocal microscope. Plan-Apochromat 20x / 0.8 N.A. objective was used. Images were captured in 16 bit.