MINSTED nanoscopy enters the Ångström localization range - StefanHellLabs
Cells of line HeLa P2, endogenously expressing NUP96 carboxy-terminally tagged with sfGFP, were grown on coverslips in high-glucose DMEM with 10% FBS and penicillin–streptomycin–amphotericin B solution . After washes in warm PBS, the cells were fixed for 30 minutes with 2.4% of freshly prepared and methanol-free formaldehyde in PBS, followed by quenching with 50 mM NHCl in PBS for 5 minutes, subsequent permeabilization with 0.
shows a COS-7 cell nucleus prepared as described above but with an additional secondary antibody, carrying STAR RED and imaged with confocal and STED.Cultured rat hippocampal neurons were prepared for MINSTED-DNA PAINT as described previously. Animal procedures were guided by the Max Planck Institute for Multidisciplinary Sciences Göttingen. Neurons were fixed , quenched for autofluorescence , permeabilized and blocked with serum proteins for 1 hour.
Before imaging, the cells were incubated with polyvinylpyrrolidone shelled silver nanoplates for 1 hour and washed with PBS.Polyvinnylpyrrolidone shelled silver nanoplates were diluted 1:500 in water, and 15 µl was dried on a coverslip that was previously cleaned with Hellmanex II and using a plasma cleaner operating with air. The coverslip was glued with double-sided scotch tape to a microscope slide to form a flow channel.
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