Synapsin Riboprobe Synthesis and In Situ Hybridization

A digoxigenin-labeled antisense riboprobe for synapsin was synthesized from cDNAs cloned in the pGEM®-T Easy Vector (Promega) following the manufacturer’s protocol (RNA labeling kit, Roche Applied Science, Monza, Italy). The length and quality of the DIG-labeled RNA synapsin probe was controlled on an agarose gel. According to manufacturers, 1 μg of probe was added to 3 volumes of RNA sample loading buffer (Sigma-Aldrich) heated to 65 °C for 10 minutes, and then chilled on ice. The probe was loaded onto a TAE/1% agarose/sybrSafe gel next to 1 kb ladder (ThermoFisher Scientific) and run at 80 V.
One band at the expected length of ≈810 nt is clearly visible (see Additional File 5a). The probe was a fragment of ~810 nucleotides complementary to the common part of long and short synapsin isoforms. In situ hybridization (ISH) was carried out on O. vulgaris SEM (see Additional File 5b), ovary and testis 20 μm-frozen sections as described by^{14 (link),52 (link)}, hybridized overnight at 60 °C. Sections were post-fixed in 4% paraformaldehyde (PFA) and mounted with Mowiol. Parallel sections were stained with hematoxylin and eosin (Sigma-Aldrich).