Fast red substrate

We followed the methods described previously (Neugebauer et al., 2009 (link)) for immunostaining. The mixed immunofluorescence and in situ hybridization technique was adapted (Thisse and Thisse, 2008 (link)) as follows: on the second day, we added the antibody anti-GFP together with the antibody Anti-Digoxigenin-AP Fab Fragments (Roche) and incubated over night at 4°C in a horizontal rotator; on the third day, we added the secondary antibody anti-rabbit Alexa Fluor 488 and incubated over night at 4°C in a horizontal rotator; on the fourth day, we developed the RNA probe with Fast-Red substrate (Roche) until a red deposit was observed and the reaction was stopped with 4% PFA (in PBS) for 5 min. her12 RNA probe was a gift from Leonor Saude. Antibodies used were anti-acetylated α-tubulin (1:300; T7451 from Sigma), anti-GFP (1:500; ab290 from Abcam), anti-DlD (1:50; zdd2 monoclonal antibody [Itoh et al., 2003 (link)]), anti-mouse Alexa Fluor 564, and anti-rabbit Alexa Fluor 488 (1:500; Invitrogen). Nuclei were stained with DAPI (1:500). Flat-mounted embryos were examined with a Zeiss LSM 710 Meta confocal microscope and a Zeiss 40x water immersion C-Apochromat lens (1.2 NA). Three-colour confocal z-series images were acquired using sequential laser excitation, and analysed using Fiji software (LSM Reader) (Schindelin et al., 2012 (link)).

Digoxigenin (DIG)-labelled RNA probes were used for in situ hybridization. For generating the Arrb2 antisense probe, mouse cDNA fragment was amplified by PCR with the antisense primer containing the T7 promoter sequence. The sequences of the primers for antisense probe were as follows:
Arrb2-F: AGAAAAACCCGGGACCAG, Arrb2-R: GATCCCCAGCACCTCCTT.
In vitro transcription was then performed from the PCR-amplified template using T7 or sp6 RNA polymerase (Roche) with Digoxigenin-UTP (Roche) for the synthesis of the antisense probe. Spinal cord sections (20 μm) were used for in situ hybridization68 (link). Pre-hybridization, hybridization and washing were performed according to standard methods. Spinal cord sections were then incubated with alkaline phosphatase-conjugated anti-Digoxigenin antibody (1:3,500; Roche) for overnight at 4 °C. After washing, the in situ signals were developed with Fast Red substrate (Roche). For further immunohistochemistry, spinal cord sections were blocked with 1% BSA for 1 h at room temperature. The sections were then incubated overnight at 4 °C with the following primary antibodies: NeuN antibody (1:1,000, mouse, Millipore, catalogue #MAB377), GFP antibody (1:500, rabbit, Abcam, catalogue #ab6556). The sections were then incubated for 1 h at room temperature with FITC-conjugated secondary antibodies (1:400; Jackson ImmunoResearch).