Figure 8

Fluorescence loss in photobleaching (FLIP) experiments demonstrate continuity of the endoplasmic reticulum in cortical explant neurones transduced with LV-pIN-KDEL. Rat cortical explant cultures were treated with LV-pIN-KDEL and imaged at 48–72 h post transduction (Materials and Methods). Images were recorded sequentially from transduced neurones and then subject to high intensity laser photobleaching at a region of interest in the soma (*, panel A, 4 minutes bleaching) or dendrite (panel site 1, 5 minutes bleaching). The beam was then switched to that used for imaging and images collected (panels B and E) and images collected. The fluorescence remaining in the cells was then quantified pre- (panels A and D) and post-bleach (panels B and E). Fluorescence intensity (F) data from the select regions of interest denoted in panels A and D, are shown in panels C and F, respectively. To facilitate the visualisation of alterations in EGFP intensity corresponding to changes in ER morphology, all images were processed using the ratio look-up table facility in Image J. Scale bar: 30 μm.