*4.9. Digital Image Analysis*

The fluorescence images were preprocessed using ImageJ. The outline of each neuronal soma was manually drawn and the quantitative analysis of the fluorescence signal was done (mean pixel intensity), as presviously described [61,62]. This quantification was carried out on both negative control (the primary antibody was omitted in the immunofluorescence protocol) and positive samples (full immunostaining protocol, including primary antibody). The average per group (control, diabetic and diabetic resistant) negative control mean intensity was subtracted from the mean intensity values of positive samples resulting the corrected mean pixel intensity. For each Hu protein, ~30 cells were scored from each mice group. Finally, we plotted the corrected mean pixel intensity calculated for each Hu protein (i.e., HuB, HuC, and HuD ) for the samples obtained from the control, diabetic and diabetic resistant mice group.
