Figure 7
Collapse assay of immunopurified P1/P2 rat retinal ganglion cells exposed to exogenous recombinant Sema3 proteins. P1 retinal ganglion cells (RGCs) were immunopurified and seeded in BD Culture Slides coated with PDL and laminin, grown overnight. The following day (effectively P2), RGCs were exposed to recombinant Sema3s for 30 minutes before fixation and immunostaining. Growth cones were considered collapsed if the F-actin did not extend past the end of the βIII-tubulin positive neurite with an arc of less approximately 60°. Only terminal growth cones were counted, because lateral extensions can occur in response to the collapse of the leading growth cone. Additionally, growth cones were only scored if their neurites were at least two soma widths long. All cultures treated with conditioned media containing recombinant Sema3 proteins showed statistically significantly higher numbers of collapsed growth cones when compared to cultures treated with control conditioned media (A). Also shown here are examples of neurons from each culture condition with growth cones scored as collapsed (hollow arrows) and uncollapsed (solid arrows). Solid arrows: uncollapsed growth cones; hollow arrows: collapsed growth cones; *** p < 0.001; scale bar: 20 μM.