Slow fade diamond mounting medium

COS-7 cells were seeded at 1 × 10⁴ cells/well in 24-well plates on glass coverslips and transfected on the next morning. After a 24 h incubation period, cells were washed with PBS and fixed 20 min using 4% paraformaldehyde and 4% sucrose in PBS at room temperature. Cells were then permeabilized with 0.15% triton X-100 in PBS for 5 min at room temperature and blocked in 10% normal goat serum (Wisent) for 20 min. Anti-GFP antibody (1:1000) was incubated 4 h at room temperature to increase the GFP signal. Cells were washed three times in 10% normal goat serum for 5 min and incubated 1 h in the dark with AlexaFluor 488-labelled goat anti-mouse secondary antibody (Invitrogen, 1:1000). Cells were washed three time in PBS, and nucleus staining was performed using 1 μg/ml Hoechst for 15 min at room temperature. Coverslips were mounted on slides with SlowFade Diamond mounting medium (Life Technologies), then confocal microscopy imaging was done using a confocal Zeiss LSM 880 2 photons microscope.

Cells were transfected for 24 h, then washed with PBS and fixed in 3% paraformaldehyde (in PBS) for 15 min. Cells were rinsed with PBS, permeabilized with 0.2% Triton-X-100 (in PBS) for 30 min, and blocked with in-situ hybridization buffer (2X SSC, 20% formamide, 0.2%, BSA, 1 µg/mL Baker’s yeast tRNA in PBS) for 15 min at 46 °C. The 5′-Cy5 oligo (dT) probe was added to the hybridization buffer at a final concentration of 10 pmol/µL and hybridized at 46 °C overnight. The oligo (dT) probe is composed of 40 thymidine labelled in 5′ with a Cy5 (IDT). The following morning, four washes were performed in the following buffer: 2X SSC, 20% formamide in water for 5 min at 37 °C; 2X SSC in water for 5 min at 37 °C; 1X SSC in water for 5 min at 25 °C and PBS for 5 min at 25 °C. DAPI (1: 10,000 in PBS) was used to stains nuclei at room temperature for 15 min. Cells were then washed two times with PBS and mounted on a slide with SlowFade Diamond mounting medium (Life Technologies, Carlsbad, CA, USA). Images were captured with a Zeiss LSM 880 2 photons confocal microscope.

Coverslips (High-precision, 1.5H, 22 ± 22 mm, 170 ± 5 mm, Marienfeld, 0107052) were immersed in 1 M KOH, sonicated for 15 min, and washed prior to incubation in poly-L-Lysine solution for 30 min. Coverslips were then washed and dried with pressurized air. Fixed cells resuspended in water were dried onto the coverslips with nitrogen and mounted on slides using Slow Fade Diamond mounting medium (Thermo Fisher, S36967). SIM was performed using a v4 DeltaVision OMX 3D-SIM system fitted with a Blaze module (Applied Precision, GE Healthcare, Issaquah, USA) using laser illumination. Images were taken in five phase shifts and three angles for each slice with Z-steps of 0.125 nm. Softworx software (GE Healthcare) was used for reconstruction with OTFs optimisation for 1.516 immersion oil.