Dig labeling mix

The cDNA clones for PVALB and for the regulator of G-protein signaling 4 (RGS4) were from the zebra finch brain cDNA ESTIMA collection, cloned in psBluescript II KS plasmids. Polymerase chain reaction (PCR) amplification was performed using forward and reverse primers with a T3 overhang (Reverse: 5’ - GTA AAA CGA CGG CCA GTG AG - 3' and Forward: 5’ - ATG ACC ATG ATT ACG CCA AG – 3’) to amplify a fragment containing the cDNA insert and T3 polymerase promoter, using the vector as template. PCR was performed in 50 µL reaction volumes containing 39.2 µL of RNase free water, 1.5 µL of 10 mM MgCl₂, 5 µL of 10x buffer, 1 µL of each primer (0.5 µM), 1 µL of dNTP mix (10 mM), 0.3 µL of Taq DNA polymerase, and 1 µL of DNA template. The temperature profile consisted of preheating at 94 °C for 3 min, 32 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 30 s, and extension at 72 °C for 90s, followed by a final extension step at 72 °C for 10 min. Then, antisense riboprobes were synthesized using T3 RNA polymerase (Lide Technologies) and DIG-labeling mix (Roche). The riboprobe reaction (Life Technologies) was performed in 20 µL reaction volumes containing 0.5 µL of RNAse inhibitor, 2 µL of DTT, 2 µL of DIG, 2 µL of 10x reaction buffer, 5 µL of Template, 2 µL of T3 enzyme mix, and 6.5 µL of nuclease-free water.

In situ hybridization expression was performed as
previously described (Jowett, 1999 (link)).
Full-length etv2 antisense probe was synthesized as previously
described (Sumanas et al., 2005 (link)). To
synthesize etv2 probe which corresponds to the 3’
portion of etv2 gene (downstream of the Gal4 integration site),
a construct containing full-length etv2 cDNA in pSPORT1 was
linearized with EciI. Antisense RNA was synthesized using SP6 RNA polymerase
(Promega) and digoxygenin (DIG) labeling mix (Roche).