Nunc lab tek 3 8 well chamber slides

SOSRS imaged before day 14 were grown on Nunc™ Lab-Tek™ III 8-well chamber slides (Thermo Scientific) and fixed in paraformaldehyde for 30 min at room temperature (RT). Older SOSRS were fixed for 1 hour in 4% paraformaldehyde in PBS in suspension at 4 °C. After 3 PBS washes, the SOSRS were incubated in 30% sucrose overnight at 4 C followed by embedding in TFM medium and freezing on dry ice. The blocks were sectioned on a Cryostat at 20 µM thickness. After 2 hours of drying at RT, the sections were washed 3 times with PBS to remove the TFM medium. For both formats, cells were permeabilized with 0.2% Triton-X 100 for 20 min at RT followed by incubation in PBS containing 5% normal goat serum with 1% BSA and 0.05% Triton-X100 for 1 h at RT. All samples were incubated in primary antibody overnight (see Table S5 for antibodies and dilutions) in the same blocking buffer at 4 C, washed 4 times in PBS with 0.05% Tween-20 (PBST), and incubated for 90 min with secondary antibody. Cells were washed 3 times in PBST and incubated with bisbenzimide for 5 min. After additional PBS washes, coverslips were mounted on slides with Glycergel mounting medium (Agilent Dako). Images were obtained on a Leica SP5 upright DMI 6000 confocal microscope.