ID-PRIME for imaging neurexin-neuroligin interactions in neuron cultures and in HEK-neuron mixed cultures.

<p>(<b>A</b>) Lipoic acid ID-PRIME labeling of pure neuron cultures. Dissociated rat hippocampal neurons were separately nucleofected at DIV0 with either 1xLAP-NLG1 plus a Venus transfection marker (shown in green), or *LplA₃₆-NRX3β plus a membrane-localized tdTomato transfection marker (shown in blue). The two pools of neurons were mixed and plated. At DIV5, neurons were labeled with lipoic acid and anti-lipoic acid antibody as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone-0052823-g004" target="_blank">Figure 4A</a>. ID-PRIME signal was detected in 22 out of 23 fields of view, and was localized to contact sites (arrow heads, row 1). Negative controls with lipoic acid omitted (row 2), AP-NLG1 in place of LAP-NLG (row 3), or with an interaction deficient mutant of NRX (row 4) are also shown. Asterisks in row 1 and 2 indicate sites where the over-expression of ID-PRIME constructs caused neuronal processes to “zip up”. (<b>B</b>) Lipoic acid ID-PRIME labeling of mixed HEK-neuron cultures. HEK cells expressing *LplA₃₆-NRX3β and a membrane-localized tdTomato marker (shown in blue) were plated on top of neurons, transfected with lipofectamine at DIV 7 with 3xLAP-NLG1 plus a Venus marker (shown in green). Labeling was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone-0052823-g004" target="_blank">Figure 4A</a>, at DIV8. ID-PRIME signal was detected in 9 out of 11 fields of view in which Venus-expressing neurons contacted Tomato-expressing HEK cells. The bottom row shows a control with a D137A mutation in NRX; no ID-PRIME signal was observed in any field of view. All scale bars, 10 µm.