Anti rabbit cy3

Intraepidermal nerve fiber (IENF) staining and quantification was performed as previously described (Geisler et al., 2016 (link)). Briefly, the footpad skin was placed in fresh picric acid fixative overnight at 4°C. The following day the samples were washed in PBS and placed into 30% sucrose for over 24 h. Samples were embedded in O.C.T. and sectioned using a cryostat in 50 micron sections and placed floating in cryoprotectant (30% sucrose and 33% ethylene glycol in PBS) and stored at −20°C. Free floating sections were then washed with PBS, blocked in 5% normal goat serum 0.3% Triton X- in PBS, then incubated in blocking solution with PGP9.5 antibody (1:1000, AB1761, Millipore) overnight at 4°C. The following day the free-floating sections were washed with PBS-T, incubated with anti-rabbit-Cy3 (Invitrogen, A10520, 1:500) for 2 h at room temperature, washed again with PBS-T, and mounted in Vectashield with DAPI.
The footpads were imaged on a Leica DMI 4000B confocal microscope using a 20× objective. Z-stacks were acquired through the whole sample and maximal projection was applied. Density was quantified by the number of PGP9.5 + axons that crossed the basement membrane and normalized to the length of the basement membrane. Densities were averaged over 3 sections per animal. Imaging and analysis were performed blinded to genotype.

Immunofluorescence was performed as previously described.³ Embryos were fixed with 90% methanol for 1 hour at −20° C, rinsed with PBS, incubated with the primary antibody overnight at 4° C and with secondary antibody for 3 hours at room temperature. Affinity purified antibodies were used at 1:50-200 for each of the anti-HsDCLK1 (Abcam #ab232875), anti-SpDCLK1 (this study), anti-SpDCLK2 (this study), anti-SP1 (Gibson & Burke, 1985) or anti-RB1 (Fernandez-Nicolas et al., 2019). Cy3 anti-rabbit (Life Technologies, ref#A10520), Alexa 488 anti-rabbit (Life Technologies, ref#A11034) or Alexa 555 anti-mouse (Life Technologies, ref#A32727) antibody was used as secondary antibody at 1:300. EdU treatment was performed at 1:1000 dilution for 30 minutes, and detection was performed by following a manufacturer’s protocol (Lifetechnologies, C10337). FITC-conjugated tubulin antibody 1:50 dilution (SIGMA, F2043) or Hoechst 33342 at a final concentration of 0.1 mg/mL (ThermoFisher, ref#62249) was used as a counter-staining. All fluorescent images were taken by confocal laser microscopy (Olympus FV3000 or Nikon W1 Spinning disk) or by fluorescence microscopy (AXIO, Vert.A1, Zeiss).