Kv2.1 clusters within the AIS are stable. Shown is a hippocampal neuron co-transfected with a 1:1 ratio of mRFP-Kv2.1 and PAGFP-Kv2.1-HA plasmid DNA. The proximal region of the single neurite expressing Kv2.1 (rectangle) was exposed to 405 nm laser light to photo-activate the PA-GFP. The top row illustrates the mRFP-Kv2.1 signal (red), the mRFP signal overlaid onto the corresponding DIC image and image acquisition under GFP optics. The middle row illustrates the PA-GFP signal immediately after photoactivation and at two representative time points (min:sec). The bottom row contains enlargements of the activated PA-GFP following photoactivation. Only small changes in cluster size and position occurred over the imaging period. There was no significant decrease in total AIS GFP fluorescence during the 42 minutes of acquisition, suggesting stable retention of Kv2.1 within the proximal axon. The imaging interval was 2 min.