Mircury lna detection probe 5 dig and 3 dig labeled

Embryos were collected at distinct embryonic stages as previously described, randing from E10-5 to E16.5, fixed for 2 h at 4 °C in 4% PFA, dehydrated in graded ethanol steps and stored at − 20 °C. Embryos were processed for whole-mount LNA-ISH was processed as previously described^{33 (link)}. miR-195 LNA-labeled microRNA probe (miRCURY LNA™ Detection probe 5′-DIG and 3′-DIG labeled, Exiqon) was used to performed whole-mount LNA ISH. For ISH on tissue sections the embryos, previously embedded in paraffin, were sectioned (10 µm) and mounted into 3-aminopropyltriethoxysilane(AAS)-coated glasses. The Mlc2a (Myl7) riboprobe was used as positive internal hybridization control, as previously reported^{34 (link)}, following the ISH protocol described by Lopez-Sanchez et al.^{35 (link)}.

Fertilized eggs (Granja Santa Isabel, Córdoba, Spain) were incubated at 38°C in forced-draft, humidified incubators. Embryos were collected at stages HH3 to HH17 (Hamburger and Hamilton, 1951 (link), 1992 (link); Lopez-Sanchez et al., 2005 (link)) and fixed overnight at 4°C in 4% PFA, dehydrated in methanol, and stored at −20°C. Embryos were processed for LNA-ISH following our previous procedure (Lopez-Sanchez et al., 2015a (link)) using miR-23b, miR-130a, miR-106a, and miR-100 LNA-labeled microRNA probes (miRCURY LNA™ Detection probe 5′-DIG and 3′-DIG labeled, Exiqon), respectively.
For histology, embryos were dehydrated with an ethanol series, cleared in isopropanol, and processed for paraplast embedding, obtaining 15-μm transverse serial sections.