Day-of-experiment protocol, including fluidics

Updated: 10/01/20

Following non-bound primary oligos wash (see FISH protocol):

Cleaning the fluidics before use (can be done a day ahead if the scope is available):

Flush water –> ~8% RBS –> water with 600 μL in reservoirs 1-15 and 3 mL in reservoirs A-D. Rate can be set to 600 μL/min. Clean again when imaging session is over. Add another last flush of 20% Ethanol.

1. Degas water in a sonicator for 5 minutes.
2. Vortex the 90 nm gold nano-urchins (GNUs), and vortex 1 μL of Tetraspeck (TS) beads in another tube. Spindown.
3. Sonicate GNUs and TS beads in separate tubes for 10 minutes.
4. Exchange the sample’s buffer to PBS.
5. When there are ~1 minute left for sonication, air-dry the sample (let it dry by itself).
6. When Sonication is done, dilute sonicated TS beads 1:250 in PBS.
7. Combine 17.5 μL 1:1 GNUs solution with 17.5 μL 1:250 TS beads solution. Final GNUs concentration is 1:2, and the final TS beads concentration is 1:500.
8. Pipette  35 μL onto the sample. Take another coverslip (same size), mark it with an ‘X’, and place it on top of the sample (sandwich it).
9. Centrifuge sample at 500 g for 3 minutes.
10. Remove ‘X’ marked coverslip. Quickly wash with PBS.
11. Wash with PBS for 3 minutes (can place on rotator).
12. Replace PBS.
13. go to the microscope, load the required expert options and fluidics protocol files.
14. Use the ‘Clean’ function (200 μL/min, 200 μL) to flush 2X SSC through all valve positions to be used in the experiment. Check that no bubbles are forming.
15. Prepare 10 mL GLOXY STORM buffer in a 15 mL falcon tube, vortex, and add 1 mL of mineral oil to prevent oxygen penetrating the buffer.
16. Prepare 10 mL wash buffer (30% formamide in 2X SSC), and 0.7 mL hybridization ‘0’.
17. Place Bioptechs stage in the microscope.
18. Lean coverslip on a Coplin jar to air-dry.
19. Assemble a Bioptechs flow cell. Low 2-holes gasket = 0.75 mm. Upper gasket = 0.5 mm thickness, new 0.5 mm thick planner gasket (~130 μL).
20. Place flow cell on stage.
21. Connect pipes to flow cells. Hold the chamber vertically when first introducing liquid into the flow cell. This can help fill the flow cell better.
22. Introduce 1300 μL of the image buffer at ~130 μL/min.
23. Once done flowing image buffer, add silicone oil onto silicone objective, and change course focus to Bioptechs preset.
24. Check the beads concentration, cell density, and noise.
25. Run super-resolution 3D calibration.
26. Run fluidics sequence 1 (wash, hybridization ‘0’, 30 minutes incubation, wash, image buffer).
27. Check signal. Calibrate VFocus.
28. If the signal is good, prepare other hybridizations.
29. Replace 2X SSC with hybridizations.
30. Choose cells. Take wide-field images.
31. Start imaging.