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.
- Degas water in a sonicator for 5 minutes.
- Vortex the 90 nm gold nano-urchins (GNUs), and vortex 1 μL of Tetraspeck (TS) beads in another tube. Spindown.
- Sonicate GNUs and TS beads in separate tubes for 10 minutes.
- Exchange the sample’s buffer to PBS.
- When there are ~1 minute left for sonication, air-dry the sample (let it dry by itself).
- When Sonication is done, dilute sonicated TS beads 1:250 in PBS.
- 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.
- 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).
- Centrifuge sample at 500 g for 3 minutes.
- Remove ‘X’ marked coverslip. Quickly wash with PBS.
- Wash with PBS for 3 minutes (can place on rotator).
- Replace PBS.
- go to the microscope, load the required expert options and fluidics protocol files.
- 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.
- 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.
- Prepare 10 mL wash buffer (30% formamide in 2X SSC), and 0.7 mL hybridization ‘0’.
- Place Bioptechs stage in the microscope.
- Lean coverslip on a Coplin jar to air-dry.
- 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).
- Place flow cell on stage.
- 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.
- Introduce 1300 μL of the image buffer at ~130 μL/min.
- Once done flowing image buffer, add silicone oil onto silicone objective, and change course focus to Bioptechs preset.
- Check the beads concentration, cell density, and noise.
- Run super-resolution 3D calibration.
- Run fluidics sequence 1 (wash, hybridization ‘0’, 30 minutes incubation, wash, image buffer).
- Check signal. Calibrate VFocus.
- If the signal is good, prepare other hybridizations.
- Replace 2X SSC with hybridizations.
- Choose cells. Take wide-field images.
- Start imaging.