Rod count and morphological parameters measured from pde6c w59 retinas with and without SchB treatment.

Pde6cw59 retinas were microdissected from 6-dpf larvae. These larvae were either exposed to DMSO carrier (pde6c DMSO) or SchB (pde6c SchB), under the same conditions that induced a positive effect on the Light-On VMR. WT retinas were included as an internal control to compare with the pde6c DMSO, as a previous study indicated a qualitative volume increase in the pde6c w59 rods [22]. All these retinas were subjected to immunostaining with the rod marker 4c12 [22] and imaged by confocal microscopy (Fig 4A–4C). Then, cell count and morphological analyses of rods were conducted in a central-retinal region on the dorsal side of the optic nerve. This mature region of the retina displayed the most prominent rod degeneration in Stearns and colleagues’ study. Analyzing this region would maximize the chance to detect morphological changes. For cell-count analysis, 4, 6, and 8 retinas were used for WT, pde6c DMSO and pde6c SchB respectively. From these retinas, rods were segmented from the 3D confocal z-stacks using parameters outlined in the methods. The subsequent morphological analyses used only those individual rods that could be successfully segmented from the images and were not distorted by their proximity to the optic nerve. The total rod numbers used in the analyses were 116, 30, and 101 for WT, pde6c DMSO and pde6c SchB respectively. The p-values less than 0.05 are highlighted in boldface. The results not only confirm that pde6c w59 rods were larger than WT rods as previously observed by Stearns and colleagues, but also reveal that the SchB treatment reduced the abnormal volume of pde6c w59 rods to a level similar to WT. Furthermore, SchB did not affect other morphological parameters and cell count of the pde6c w59 rods. The raw data for Table 2 are available in S3 File.