A DNA fragment encoding part of Papp-aa sequence was amplified using primers in Supplementary file 1 . The PCR product was cloned in pGEM-T easy plasmid. The Digoxygenin-UTP labeled sense and antisense riboprobes were synthesized as previously reported (Wang et al., 2009 (link)). Zebrafish larvae were fixed in 4% paraformaldehyde, permeabilized in methanol, and analyzed by whole mount immunostaining or in situ hybridization analysis as described previously (Dai et al., 2014 (link)).
For double color in situ hybridization and immunostaining, papp-aa mRNA signal was detected using an anti-DIG-POD antibody (Roche), followed by Alexa 488 Tyramide Signal Amplification (Invitrogen). After in situ hybridization analysis, the stained larvae were washed in 1xPBST then incubated with a GFP antibody overnight at 4°C. Larvae were then stained with a Cy3 conjugated Goat anti-Rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA). Images were acquired using a Nikon Eclipse E600 Fluorescence Microscope with PMCapture Pro six software.
For double color in situ hybridization and immunostaining, papp-aa mRNA signal was detected using an anti-DIG-POD antibody (Roche), followed by Alexa 488 Tyramide Signal Amplification (Invitrogen). After in situ hybridization analysis, the stained larvae were washed in 1xPBST then incubated with a GFP antibody overnight at 4°C. Larvae were then stained with a Cy3 conjugated Goat anti-Rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA). Images were acquired using a Nikon Eclipse E600 Fluorescence Microscope with PMCapture Pro six software.