Figure 3

Sema5B expression in hippocampal neurons. (a-r) Confocal images of 7 to 10 days in vitro (DIV) hippocampal cultures immunolabeled with anti-5B antibody plus the specified marker. (a-c) Immunolabeling of a hippocampal neuron transfected with green fluorescent protein (GFP)-Sema5B demonstrated that Sema5B levels were markedly higher in the GFP-Sema5B-expresing cells (arrow) compared to the surrounding, untransfected neurons. (d-i) In untransfected cells, Sema5B was expressed in dendrites labeled with microtubule-associated protein 2 (MAP-2) (arrows, (d-f)) and axons labeled with tau (arrowheads; g-i). (j-l) Both glutamate decarboxylase (GAD)-65-positive (arrowhead) and GAD-65-negative (arrow) cells expressed Sema5B. (m-o) Coimmunolabeling with the postsynaptic marker, postsynaptic density (PSD)-95, demonstrated minimal colocalization (insert, arrows), demonstrating that Sema5B is not particularly enriched at synapses. (p-r) Sema5B expression was also observed in glial fibrillary acidic protein (GFAP)-positive glial cells. Scale bar; a-l 40 μm, m-o 20 μm and p-r 40 μm.