3.1. Neuron growth and transfection
To obtain neurons with mature axon and dendritic structure, primary
hippocampal neurons were isolated and cultured for 5 days (Figure S1).
Immunofluorescence assay revealed that the neurons were all
Tuj1-positive and possessed a mature axon of approximate 0.74 μm width
(Figure S1A). After that, the Tuj1-positive neurons were transfected to
express mCherry proteins by a plasmid encoding CMV promoter-driven
mCherry gene (Figure 1A). A continuous fluorescence imaging of a single
neuron found that mCherry protein could be detected and evenly
distributed in the cell soma and axon of neurons at 48 h after
transfection (Figure 1B and Figure S1B). And then the clear granular
fluorescence signal was visible between 60 h and 72 h, suggesting the
formation of mCherry aggregations during this period. Finally, the
fluorescence signal of the whole neuronal axon became discrete at 78 h.
These results suggested an optimal axonal transport observing window
between 60 h and 72 h after transfection, considering that the
over-expression of mCherry protein for more than 72 h would affect the
physiological state of neurons. It also demonstrated that the sparse
transfection strategy can serve as a facile strategy to produce and
image fluorescent protein aggregates in a single living neuron.