Figure 1. Cortical cells in long-term culture and in old rat brains had higher SA-β-gal activity and accumulated lipofuscin. (A) SA-β-gal activity or lipofuscin accumulation detected by autofluorescence or by Sudan Black B (SBB) staining were detected in primary rat cortical cells cultured for the indicated DIV. Notice that cortical cells have higher SA-β-gal activity and lipofuscin from 26 DIV. Images are representative of at least three independent experiments. Scales bar represent 100 μm. (B) Percentage of SA-β-gal or SBB positive cells in the cultures incubated at the indicated DIV. Quantification was made using NIS Elements software. The mean of three independent experiments, each done by quintupled replicas, is graphed. Bars in graphs represent SEM. Two-way RM ANOVA analysis, with Dunnett´s multiple comparison test. **** p< 0.0001 in comparison with 6 DIV. (C) Cortical neurons in old brains had higher SA-β-gal activity (scale bars represent 500 μm) and accumulated lipofuscin. Scale bar represents 15 μm. (D) The percentage of SA-β-gal positive area within each brain section is plot. The average of three brains per age is graphed; 15 sections from each brain were quantified. Bars in graphs represent SEM. Unpaired t Test, ** p< 0.01. (E) Quantification of autofluorescence intensity per section (arbitrary units). Bars in graphs represent SD. The average of three brains per age is graphed; 15 sections from each brain were quantified. Even though there was an evident increase in autofluorescence, no statistical significance was obtained.