Computational protocol: Viral Vector Based Improvement of Optic Nerve Regeneration: Characterization of Individual Axons’ Growth Patterns and Synaptogenesis in a Visual Target

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Protocol publication

[…] The mean PTEN staining intensity in RGCs was measured using ImageJ software. Values for PTEN were based on 8 cells per section, 2 sections per case, and 3 per groups. Only GFP+ cells in the ganglion cell layer with visible axon projection (i.e. RGCs) were selected for measurement. As negative controls, we omitted the primary antibody. Individual mean values were corrected by staining levels in the corresponding negative controls (i.e. background subtracted) and then averaged across each group. [...] LSFM was performed as previously described , . For each optic nerve and brain between 100 and 500 optical slices were imaged with LSFM (LaVision) at a scan speed of 0.5–1.5 s per slice. Images were collected at 2 to 5μm increments in the Z axis. Images, and 3D volume rendering were prepared using Imaris software v7.6.1 (Bitplane). CTB-labeled RGC axons in the brain were traced using Imaris Filament Tracer Module and SCN boundaries were marked with Imaris Surface Module. Axons were identified and manually traced in individual 2D optical slices (z stacks) and compiled to give a 3D reconstruction. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications SPIM, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Wounds and Injuries, Optic Nerve Injuries