title: A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining.
abstract: Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.
authors.forename: Nicole
authors.initials: N
authors.lastname: Staudt
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.
authors.forename: Nicole
authors.initials: N
authors.lastname: Müller-Sienerth
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.
authors.forename: Alla
authors.initials: A
authors.lastname: Fane-Dremucheva
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.
authors.forename: Shahnaz P
authors.initials: SP
authors.lastname: Yusaf
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.
authors.forename: David
authors.initials: D
authors.lastname: Millrine
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK. Electronic address: gw2@sanger.ac.uk.
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Wright
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000941:Antigens / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D007150:Immunohistochemistry; D051379:Mice; D009474:Neurons / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D015027:Zebrafish
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D011506:Proteins; D011994:Recombinant Proteins
keywords: Immunohistochemistry; Monoclonal antibodies; Neuron; Receptor; Zebrafish