Double-label In Situ Hybridization of Axon Guidance and Odorant Receptor Genes

Double-label in situ hybridization was performed using antisense digoxigenin (DIG) RNA probes to guidance genes and fluorescein-labeled probes to OR subfamily genes as previously described [36 (link), 64 (link)], with the exception that RNase treatment was not performed after probe removal. Following probe hybridization and removal, embryos were incubated in anti-DIG-POD (1:500; Roche, 11207733910) and the DIG label was amplified using the cyanine 5-coupled tyramide kit to label axon guidance genes. OR transcripts were detected using anti-fluorescein-POD (1:500; Roche, 11426346910) and the fluorescein label was amplified using a fluorescein-coupled tyramide kit (PerkinElmer, NEL741001KT). Propidium iodide labeling and imaging were performed following the second tyramide amplification [75 ]. The plasmids used to make probes targeting nrp1a and nrp1b were as described by Dell et al., 2013 [76 (link)] and Taku et al., 2016 [64 (link)]. For robo2 (refseq accession number NM_131633.1, nucleotides 3445–4382) and pcdh11 (refseq accession number XM_005173190.3 nucleotides 220–839) sequences were amplified from 48hpf zebrafish cDNA and cloned into pcRII-TOPO Dual promoter TA cloning kit (Invitrogen, K460001) for probe synthesis. Full-length probes were used in all hybridization experiments except for robo2 where the probe was hydrolyzed prior to incubation.