Figure 1

Preparation and live imaging of postnatal mouse retinal explants. (A) Schematic representations of upright and inverted imaging preparations. (B) Retinal and imaging orientations for use with cellular movement and morphology analysis. P = peripheral retina; C = central retina; A = apical neuroblastic margin; B = basal neuroblastic margin. (C) An example of conventional histological data in which thymidine analogue (CldU for 30 minutes at 29.5h post-electroporation) immunolabeling provides a means to evaluate the position and morphology of s-phase and non-s-phase retinal cells expressing HuSH-scrambled shRNA-tGFP. NBL = neuroblastic layer; GCL = ganglion cell layer. (D) An example of live 2-photon imaging in retinas transfected with H2B-GFP at P0, and imaged 20h later. (E) Live, 2-channel confocal imaging of retinas co-transfected at P0 with H2B-GFP (300 ng/μl), a trace amount (50 ng/μl) of CMV-Cre, and the Cre-sensitive reporter pCALNL-dsRed (500 ng/μl; final ratio of 6:1:10). Images were acquired 48h post-transfection. (F) High magnification insets (red box in E). White arrowheads indicate variability in terminal nuclear position; magenta arrowheads indicate morphological features of cytoplasmic dsRed localization in apical processes.