Cloning of the 5F11 monoclonal antibody. (a) Reverse transcriptase-polymerase chain reaction (PCR) amplification of the variable light (V_Lκ) and heavy (V_H) antibody chains from the 5F11 hybridoma. (b) Assembly of the 5F11 V_Lκ and V_H regions by fusion PCR through a joining fragment resulting in a 2.4 kbp product. (c) Schematic of the plasmid encoding the recombinant antibody with the fusion PCR product cloned between NotI and AscI. (d) Western blot run under reducing (left panel) or non-reducing (right panel) conditions: tissue culture medium (Med), supernatant of cells producing the recombinant 5F11 antibody (Rec) or tissue culture medium spiked with the 5F11 monoclonal antibody purified from the parent hybridoma (Hyb). Staining of the recombinant 5F11 [Rec 5F11, (e)] is indistinguishable from that of the parent hybridoma [Hyb 5F11, (f)], red. Nuclei are counterstained in blue (DAPI). Abbreviations: cns = central nervous system; e = eye; CMV = cytomegalovirus promoter; L = leader sequence; C = constant region; V = variable region.

Recombinant monoclonal antibodies recapitulate their gene expression patterns on wholemount fixed zebrafish embryos. (a-c) Dorsal views of a 24 hpf zebrafish embryo showing flrt3 gene expression by in situ hybridization on the midbrain side of the mid-hindbrain boundary (a) and antibody staining with SI3-Flrt3 [green in (b), white in (c)]. (d-f) Lateral view of a dissected 24 hpf eye showing expression of the unc5b gene in the dorsal retina (d) and antibody staining with SI2-Unc5b [green in (e), white in (f)]. (b, e) red = DAPI nuclear stain. Scale bars = 50 μm.