Longitudinal assessment of fluorescence stability shows fluorescence intensity decreases over time: implications for fluorescence microscopy studies
Fig 9
AF546-conjugated secondary antibody is most resistant to decreases in fluorescence intensity over time while AF647-conjugated secondary antibody is least resistant.
Control Experiment 1: all sections were stained with anti-OMP primary antibody, followed by staining with AF488-, AF546- or AF647-conjugated secondary antibody and underwent widefield imaging daily for a week. A two-way repeated measures ANOVA showed significant effects of fluorophore (F(2,6)=161.7, p < 0.001) and time point (F(2.44,14.7)=152.5, p < 0.001) and a significant interaction between fluorophore and time point (F(4.89,14.7)=68.0, p < 0.001). (a-c) Images of AF488-conjugated secondary antibody-stained section. (d) Significant decrease in fluorescence intensity across one week of daily imaging for AF488-conjugated secondary antibody. Note that these are the same data as shown in Fig 1g. (e-g) Images of AF546-conjugated secondary antibody-stained section. (h.) No change in fluorescence intensity across one week of daily imaging for AF546-conjugated secondary antibody. (i-k) Images of AF647-conjugated secondary antibody-stained section. (l) Significant decrease in fluorescence intensity across one week of daily imaging for AF647-conjugated secondary antibody. Images of days 1, 4 and 7 are shown as examples. Each line of data represents a single mouse.