Densitometric analysis of fluorescence images

Densitometric analysis of LAMP1 and LC3 proteins was performed on perfused rat brain sections. In order to avoid staining variability among sections and experimental groups, sections of rat brains of the different experimental groups were incubated with the appropriate primary and secondary antibodies at the same time. Furthermore, confocal settings for image capture were maintained constant throughout the acquisition of sections from the different experimental groups.

After background subtraction, LAMP1- or LC3- associated signals were quantified by manually outlining individual RN positive neurons and measuring cell-associated fluorescence intensity with the ImageJ software [38]. The F/A ratio defines mean fluorescence of individual cells (F) normalized to the total cellular surface (A). Quantification was done on 30 cells per animal (n = 30 cells/rat; N = 5 rats/group).

For TFEB subcellular localization (cytoplasm/nucleus) quantification was performed using five alternate sections of 30-mm regularly spaced throughout the entire RN rostrocaudal extension (n = 5 sections/rat; N = 5 rats/group). The process was made off-line and only neurons identified by a clear nuclear profile were included for analysis and cells presenting nuclear TFEB expression were expressed as percentage of the total number of RN neurons.

Data collecting for densitometry were done by the experimenter blind to the group analyzed.