Immunohistochemistry of the embryonic zebrafish brain with monoclonal antibodies to neural receptors and secreted proteins. The embryo stage and transcript/protein are indicated within each subpanel and staining patterns described within the main text. Transcript localisation is shown by wholemount in situ hybridisation as brightfield images; images of antibody staining are taken by confocal microscopy (red); nuclei are stained with DAPI in G^II (green). Note that some antibody staining, for example Igsf21a in the ventral diencephalon (B^I), is outside of the optical plane. Orientation of the embryos is always anterior to the left with ventral views in A, A^I, D^I, I^I and J^I; lateral in A^II, C, C^I, D, D^II, D^III, E, E^I, G, G^I, H, H^I, J and J^III; and dorsal in A^III,B, B^I, F, F^I, G^II, G^III, I and J^II. Scale bars represent 100 µm in C^I and E^I, for all other images 50 µm. Abbreviations used in all figures are: ac = anterior commissure, dvdt = dorso–ventral diencephalic tract, epi = epithalamus, hind = hindbrain, hypo = hypothalamus, MHB = mid-hindbrain boundary, mid = midbrain, ob: olfactory bulb, oe = olfactory epithelium, poc = postoptic commissure, sot = supraoptic tract, tec = tectum, teg = tegmentum, tel = telencephalon, th = thalamus, tpc = tract of the posterior commissure, tpoc = tract of the postoptic commissure, vlt = ventral longitudinal tract, vnc =  ventral nerve cord, hpf = hours post-fertilisation, dpf = days post-fertilisation.

Antibodies detect neural receptor proteins within discrete subcellular localisations. (A) Anti-Sema4c staining in the anterior neuroepithelium. (B) Anti-Kita labels the anterior (ac) and postoptic (poc) commissures and is also enriched at the periphery of cells (arrow). (C) Anti-Kita also stained axons of the ventral nerve cord (vnc) and the surface of hindbrain cells. Anti-CD276 staining in the developing midbrain (D), and tectum (E). (F) Anti-Igsf21b stained axon-rich regions in the anterior forebrain where it was often concentrated in discrete puncta (arrow). Views are anterior to the left and dorsal for A and E, lateral for B, C and F, ventral for D. Scale bars represent 100 µm in (A) and 50 µm in (B–F).

The addition of different tags facilitates multiplex staining. (A) Schematics showing the addition of C-terminal Bio-6-His and FLAG tags to plasmids. The region encoding the specific antibodies are flanked by unique NotI and AscI sites facilitating antibody tag switching by subcloning. (B–D) The same 5 dpf embryo was simultaneously stained with both the biotin-tagged anti-Lrrc3b and FLAG-tagged anti-Sema4c antibodies. The staining of anti-Lrrc3b in green (B) and anti-Sema4c in red (C), together with the merged images (D) at the same site are shown. Staining patterns are described in the main text. Scale bars represent 50 µm.