Nbt bcip

The cDNAs used as templates to make probes were prepared by RNA extraction and reverse transcription PCR (RT-PCR). The primers for RT-PCR were designed according to the Esembl genomic sequences [19 (link)]. In situ hybridization was performed as described previously [20 (link)] with minor modifications. Briefly, DIG-labeled riboprobes were incubated with the embryos to detect the expression pattern of CRMP2 and CRMP4. Anti-DIG-alkaline phosphatase Fab fragments (Roche, Mannheim, Germany) and NBT/BCIP (Sangon Biotech, Shanghai, China) were used to detect and amplify the signals.

For western blot detection, samples were taken and weighed, followed by ground in liquid nitrogen. Two volumes of 1 × SDS loading buffer was added and boiled for 10 min. After centrifugation for 10 min at 12 000 rpm, 20 μl supernatant was loaded per lane on the SDS-PAGE gel. Then proteins were transferred to the nitrocellulose membranes. The secondary antibody was purchased from Sigma-Aldrich, Inc., Alkaline phosphatase was visualized with NBT/BCIP (Sangon Biotech, Shanghai, China).

The experiment of whole-mount in situ hybridization was performed as described by Thisse and Thisse (2008 (link)). Fish embryos at different developmental stages were fixed in 4% paraformaldehyde/PBS at 4°C overnight, and then dehydrated in methanol with a gradient from 25 to 100% for storage. When the fixed embryos were analyzed, they were gradiently rehydrated to 0.1% PBST (0.1% Tween-20 in phosphate-buffered saline). Embryos at different developmental stages (24 hpf to 72 hpf) were digested with proteinase K from 1 μg/ml to 80 μg/μl for 6–30 min based on hpf. The treated embryos were then incubated in digoxin-labeled probes for hybridization at 70°C overnight. The embryos were transferred to the preheated 50% SSCT-50% hybridization buffer for 30 min at 70°C, blocked in 10% sheep serum in 0.1% PBST, then incubated with anti-DIG antibody at 4°C overnight. The embryos were then incubated in NBT/BCIP (Sangon Biotech, Shanghai, China) staining solution for visualizing stained signals. Embryos were mounted in methylcellulose and were scanned using a Nikon AZ100 microscope with Nikon Digital Sight DS-Fill digital camera (Nikon, Japan). Images were captured and processed with NIS-Elements F 3.0 (Nikon).