Pannoramic confocal scanner

The immunofluorescence (IF) detection of ferritin was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center. Automated IF staining was conducted using Leica Bond BX staining system. Cells cultured on chamber slides were fixed in 4 % paraformaldehyde (PFA, Electron Microscopy Sciences, 157–8–100) for 15 min followed by washing in PBS. Slides were loaded in Leica Bond and IF staining was performed as follows. The primary antibodies against FTH1 (Rb, 0.11μg/mL, Cell signaling technology, 4393) were incubated with the sample for 1 hour at room temperature. After that, the Leica Bond Polymer anti-rabbit HRP secondary antibody (included in Polymer Refine Detection Kit (Leica, DS9800)) was used for 8 min, followed by incubation with Alexa Fluor tyramide signal amplification reagents (Life Technologies, B40953) for 10 min. After the run was finished, slides were washed in PBS and incubated in 5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, D9542) in PBS for 5 min, rinsed in PBS, and mounted in Mowiol 4–88 (Calbiochem). Slides were kept overnight at - 20°C before imaging. The slides were scanned with a 20x/0.8NA objective on a Pannoramic Confocal Scanner (3DHistech, Budapest, Hungary).