Prolong gold dapi mounting media

After optical redox imaging, cells were fixed with 2% paraformaldehyde for 15min and then washed once with PBS before storage in PBS overnight. The next day immunofluorescence staining was performed as previously described.⁴⁴ (link) Conjugated antibodies against Ki67 and CC3 (11882S-488 conjugate, and 8172S-594 conjugate, Cell Signaling Technology, Danvers, Massachusetts) both at a dilution of 1:50 were applied before coverslips were mounted onto slides with Prolong Gold DAPI mounting media (P36931, Invitrogen, Carlsbad, California) and sealed with clear nail polish. After 24 h stored at 4°C in a dark box, the slides were imaged on a Nikon Eclipse Ti2 inverted widefield fluorescence microscope using a $20\times$ air objective (0.75 NA), with standard Nikon filters for DAPI, FITC, and Texas Red [DAPI: ex: $375/28\mathrm{nm}$ , em: $460/60\mathrm{nm}$ ; FITC: ex: $480/30\mathrm{nm}$ , em: $535/45\mathrm{nm}$ ; Texas Red: ex: $560/40\mathrm{nm}$ , and em: $630/60\mathrm{nm}$ ). For analysis, six FOVs were acquired from two separate biological replicates for a total of 12 FOVs analyzed per condition. Co-localization analysis was performed using a previously described CellProfiler pipeline²⁶ (link) that identifies DAPI, Ki67, and CC3 objects. The number of DAPI objects that overlapped with Ki67 or CC3 positive stain were counted to calculate the percent of total cells positive for Ki67 or CC3.