Quantitative Analysis of Connexin Plaques

All cell counting (cells/mm²) was executed by two different, blind to the experiment investigators, on six areas per section (which contained GFP⁺ cells) spaced at least 50 μm apart. Only Dapi⁺ and BrdU⁺ nuclei were taken into account. Images were captured using an Axioplan‐2 optical/widefield fluorescent (Zeiss) while confocal images were captured with Nikon Eclipse Ti and were assembled in ImageJ (Fiji) software. Demyelination, axonal loss, and inflammatory cell evaluation were analytically described in our previous work [27 (link)]. Connexin immunoreactivity was quantified with Bitplane Imaris software (Oxford Instruments) from analysis of multidimensional microscopy datasets. 3D reconstruction and visualization of the cells and connexins were rendered through the program from multiple z stacks. GJ plaques (Cx32 and Cx47) were defined as a concentration of connexin signal with size limits between 0.1 and 1 μm² [28 (link)]. The total number of connexin GJ plaques (Cx32 and Cx47) was measured in each image and then a ratio to the respective cell (BrdU⁺) was calculated, as previously described [29 (link)].