Combinatorial mRNA Riboprobe and Immunohistochemistry

mRNA riboprobes were obtained by PCR amplification using primers designed from NCBI sequences of each gene. cDNA was cloned into the pCRII TOPO vector (Invitrogen) and linearized DNA was transcribed to generate digoxigenin labeled sense and antisense riboprobes. These riboprobes were used for whole mount in situ hybridization on chick embryos as previously described (Ratié et al., 2013 (link)).
After in situ hybridization, embryos were dehydrated with methanol overnight to improve permeability of the antibody for immunohistochemistry (Lumsden and Keynes, 1989 (link)). Anti-HuC/D mouse (1:500; molecular probes; A21271) primary antibody was used and detected with a peroxidase-conjugated rabbit-anti-mouse secondary antibody (1:2000; Jackson ImmunoResearch; 315-035-045).