<p>MeWo-GFP cells (green) infected with cell-free VZV66RFP (red) virus were plated into axonal compartments of microfluidic chambers or in glass-bottom culture dishes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126081#pone.0126081.g001" target="_blank">Fig 1C</a>). Cultures were fixed and immunostained for the medium neurofilament subunit (NF-M, white) 1 to 3 dpi and nuclei stained with Hoechst (blue). (A–D) Control experiments with uninfected MeWo-GFP cells. No GFP was found in the NF-M+ axons. (E–H) Immunostaining of an axonal compartment where MeWo-GFP cells infected with VZV66RFP were plated 3 days prior to fixation. (E&F) Images showing GFP and ORF66RFP fluorescence, respectively. The arrow points to a syncytium of MeWo cells containing both fluorescent proteins. Arrowheads point to axons showing GFP and RFP signal. (G) shows immunocytochemical staining of this field for NF-M, the syncytium observed in E and F to contain both GFP and RFP is NF-M+ as well (white). H shows a merge of all channels, where both white (NF-M) and yellow (co-expression of GFP and RFP) staining are present in the same polykaryon. (I–L) An axonal compartment with VZV-GFP-infected ARPE cells (green) was immunostained at 4 dpi with anti-NF-H 4142 (red) and anti-VZV gI (white) antibodies. I shows GFP fluorescence and J, the immunostaining for NF-M. Many GFP+/NF-M+ axons (arrowheads) were observed. Some infected ARPE cells (arrow) became neurofilament+ after fusion with axons. The fused ARPE cells and axons were also immunopositive for glycoprotein I (K), but not several other cells that were not part of the polykaryon. Scale bar: 50μm.</p
