Table 1.
Summary table of primary and secondary antibodies used. Species that antibody was raised in, dilution used, incubation time, manufacturer, and catalog number are provided.
Table 2.
Summary of widefield imaging parameters. Combination of primary antibody and fluorophore is provided. Revolve widefield microscope (Echo) imaging parameters are also provided including digital haze reduction, gain, exposure, and LED power.
Table 3.
Summary of confocal imaging parameters. Combination of primary antibody and secondary antibody fluorophore is provided. Confocal imaging parameters provided, including laser power, gain, sampling speed, and pixel size.
Fig 1.
Breakdown of analyses completed for each experiment using Control Exp. 1 (anti-OMP primary, AF488-conjugated secondary) as an example.
(a-c) Example images of OB section used for Control Exp. 1 (anti-OMP-AF488). (a) Image without overlays. (b) Same image with overlaid ROI used to assess combined background + staining fluorescence. (c) Same image with overlaid ROI used to assess background fluorescence (i.e., external plexiform layer (EPL)), in which no OMP staining is present. (d) Change in raw mean gray fluorescence values for total, i.e., background + stain (circle), stain (square), and background (triangle). (e) Percentage normalized mean gray fluorescence for total (combined background and stain) fluorescence. (f) Percentage normalized mean gray fluorescence for background fluorescence. (g) Percentage normalized mean gray fluorescence for staining fluorescence signal (total – background). Each line of data represents a single mouse.
Table 4.
Summary of statistical results for each set of analyses comparing fluorescence intensity over time. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; NS (non-significant) = p ≥ 0.05. d.f.: degrees of freedom.
Fig 2.
Anti-OMP-AF488 and anti-HA-AF546 stained OE sections show significantly decreased fluorescence intensity over the course of weekly widefield imaging.
(a-c) Anti-OMP primary antibody with AF488 secondary antibody-stained section imaged with widefield microscopy. (d) Anti-OMP primary antibody staining combined with AF488-conjugated secondary antibody staining of OE sections show significant decreases in fluorescence intensity. (e-g) Anti-HA primary antibody-stained with AF546 secondary antibody-stained section imaged with widefield microscopy. (h) Anti-HA primary antibody staining combined with AF546-conjugated secondary antibody staining of OE sections show significant decreases in fluorescence intensity. Images of weeks 1, 4 and 7 are shown as examples. Each line of data represents a single mouse.
Fig 3.
Decreased fluorescence intensity of OE sections triple-stained for OMP-AF488, GAP43-AF546, and EdU-sulfo-Cyanine5 over the course of weekly widefield fluorescence microscopy.
Sections were triple-stained with anti-OMP primary/AF488-conjugated secondary, anti-GAP43 primary/AF546-conjugated secondary and EdU (click chemistry detection with sulfo-Cyanine5). (a-c) OMP-stained OE section. (d) Significant decrease in fluorescence intensity for OMP-stained OE over time. (e-g) GAP43-staining in the same OE section shown in a-c. (h) Significant decrease in fluorescence intensity for GAP43-stained OE over time. (i-k) EdU staining in the same OE section shown in a-c and e-g. (l) Trend towards a decrease in fluorescence for EdU-stained OE over time. Images of week 1, week 4, and week 7 are shown as examples. Each line of data represents an individual mouse.
Fig 4.
Decreased fluorescence intensity of OE sections triple-stained for OMP-AF488, GAP43-AF546, and EdU-sulfo-Cyanine5 over the course of weekly confocal microscopy.
Sections were triple-stained with anti-OMP primary/AF488-conjugated secondary, anti-GAP43 primary/AF546-conjugated secondary and EdU (click chemistry detection with sulfo-Cyanine5). (a-c) MIPs of anti-OMP primary antibody with AF488-conjugated secondary antibody-stained OE section imaged with confocal microscopy. (d) Significant decrease in AF488 fluorescence intensity in single optical sections. (e) Significant decrease in AF488 fluorescence intensity in MIPs. (f-h) MIPs of anti-GAP43 primary antibody with AF546-conjugated secondary antibody-stained OE section imaged with confocal microscopy. (i) Significant decrease in AF546 fluorescence intensity in single optical sections. (j) Significant decrease in AF546 fluorescence intensity in MIPs. (k-m) MIPs of EdU with sulfo-Cyanine5 fluorophore-stained OE section imaged with confocal microscopy. (n) Significant decrease in sulfo-Cyanine5 fluorescence intensity in single optical sections. (o) Significant decrease in sulfo-Cyanine5 fluorescence intensity in MIPs. Images of weeks 1, 3 and 5 are shown as examples. Each line of data represents an individual mouse.
Fig 5.
OMP-AF488 stained OB sections showed a significant decrease in fluorescence intensity over time.
(a-c) Anti-OMP primary antibody and AF488-conjugated secondary antibody-stained OB section imaged with widefield microscopy. (d) OB sections imaged weekly show a significant decrease in fluorescence intensity. Images of weeks 1, 4 and 7 are shown as examples. Each line of data represents a single mouse.
Fig 6.
Decrease in fluorescence intensity over time is dependent on secondary antibody fluorophore.
(a-c) Anti-GAP43 primary antibody and AF488-conjugated secondary antibody-stained OB section imaged with widefield microscopy. (d) Anti-GAP43-AF488 staining in GL shows a significant decrease in fluorescence intensity. (e) Anti-GAP43-AF488 staining in GCL shows a significant decrease in fluorescence intensity. (f-h) Anti-GAP43 primary antibody and AF546-conjugated secondary antibody-stained OB section imaged with widefield microscopy. (i) Anti-GAP43-AF546 staining in GL shows no significant change in fluorescence intensity. (j) Anti-GAP43-AF546 staining in GCL shows no significant change in fluorescence intensity. (k-m) Anti-GAP43 primary antibody and AF647-conjugated secondary antibody-stained OB section imaged with widefield microscopy. (n) Anti-GAP43-AF647 staining in GCL shows a significant decrease in fluorescence intensity. (o) Anti-GAP43-AF647 staining in GCL shows a significant decrease in fluorescence intensity. Images from weeks 1, 4 and 7 are shown as examples. Each line represents data from a single mouse.
Fig 7.
Fluorescence intensity changes over time in OB dopaminergic neurons.
(a-c) Chicken anti-TH primary antibody and AF488-conjugated secondary antibody-stained OB section imaged with widefield microscopy. (d) OB sections stained with chicken anti-TH primary antibody and AF488-conjugated secondary antibody show a significant decrease in fluorescence intensity. (e-g) Rabbit anti-TH primary antibody and AF488-conjugated secondary antibody-stained OB section imaged with widefield microscopy. (h) OB sections stained with rabbit anti-TH primary antibody and AF488-conjugated secondary antibody show a trend towards a decrease in fluorescence intensity that does not reach significance. (i-k) GCaMP6s fluorescence in OB dopaminergic neurons over time imaged with widefield microscopy. (l) OB sections imaged weekly for GCaMP6s fluorescence show significant decrease in fluorescence intensity. Images of weeks 1, 4 and 7 are shown as examples. Each line of data represents a single mouse.
Fig 8.
Fluorescence intensity of Iba1 staining in cortex and hippocampus decreases over time.
(a-c) Anti-Iba1 primary antibody and AF546-conjugated secondary antibody-stained cortex layer 1 imaged with widefield microscopy. (d) Cortical sections stained with Iba1 primary antibody and AF546-conjugated secondary antibody show a trend towards a significant decrease in fluorescence intensity. (e-g) Anti-Iba1 primary antibody and AF546 conjugated secondary antibody-stained hippocampal dentate gyrus and Cornu Ammonis 1 subregions imaged with widefield microscopy. (h) Results for hippocampal sections stained with Iba1 primary antibody and AF546-conjugated secondary antibody show a significant decrease in fluorescence intensity. Images of weeks 1, 4 and 7 are shown as examples. Each line of data represents a single mouse.
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.
Fig 10.
AF546 is the most resistant to fluorescence intensity changes over time and photobleaching does not underlie the observed changes in fluorescence intensity.
All sections were stained with anti-OMP primary antibody, followed by staining with AF488-, AF546- or AF647-conjugated secondary antibody. Control Experiment 2 (a-i) consisted of an initial imaging session followed by a second imaging session 7 weeks later, using widefield microscopy. A two-way repeated measures ANOVA showed significant effects of fluorophore (F(2,6)=317.3, p < 0.001) and time point (F(1,6)=747.5, p < 0.001), and a significant interaction between fluorophore and time point (F(2,6)=317.3, p < 0.001). Note that some data points have similar values and overlap (n = 3). (a,b) Images of AF488-conjugated secondary antibody-stained section at weeks 1 and 7. (c) No significant difference in fluorescence intensity after six weeks without imaging for AF488-conjugated secondary antibody-stained sections. (d,e) Images of AF546-conjugated secondary antibody-stained section at weeks 1 and 7. (f) No significant difference in fluorescence intensity after six weeks without imaging for AF546-conjugated secondary antibody-stained sections. (g,h) Images of AF647-conjugated secondary antibody-stained section at weeks 1 and 7. (i) No significant difference in fluorescence intensity after six weeks without imaging for AF647-conjugated secondary antibody-stained sections. Control Experiment 3 (j-u) consisted of widefield imaging every two minutes, collecting a total of 7 data points across 13 minutes. A two-way repeated measures ANOVA showed significant effects of fluorophore (F(2,6)=5.79, p = 0.040) and time (F(2.16,13.0)=12.63, p < 0.001) but no significant interaction between fluorophore and time (F(4.32,13.0)=2.95, p = 0.059). (j-l) Images of AF488-conjugated secondary antibody-stained section at minutes 1, 7 and 13. (m) No change in fluorescence intensity for AF488-conjugated secondary antibody-stained sections imaged every two minutes. (n-p) Images of AF546-conjugated secondary antibody-stained section at minutes 1, 7 and 13. (q) No change in fluorescence intensity for AF546-conjugated secondary antibody-stained sections imaged every two minutes. (r-t) Images of AF647-conjugated secondary antibody-stained section at minutes 1, 7 and 13. (u) No change in fluorescence intensity for AF647-conjugated secondary antibody-stained sections imaged every two minutes. Each line of data represents a single mouse.