For STORM I’m using 35 mm MatTek dishes. Their surface area is ~8 times smaller than 100 mm dish. If I’m splitting IMR90 1:5, then I’m taking 1 volume of that and diluting 1:8, in total, a dilution of 1:40 and then seed 8 35 mm dishes, 2 ml each. Once it’s confluent, I fix and permeabilize.
For seeding without having to grow the cells on the MatTek dishes, I first clean and cover the glass-bottom with Poly-l-lysine. Following trypsinization, I resuspend the cells in 4 ml, and pipette 100 ul into each glass-bottom, so 1:40 dilution, then incubate at 37c for ~4 hours.
*** try splitting 1:5, dispense 100 ul into each plate and incubate overnight (NO poly-l-lysine), and fix next day.
Fixation and permeabilization protocol (Adapted from Sonny) 9/22/15
- Trypsinize a confluent 100 mm dish, centrifuge (1200 rpm for IMR90) and resuspend in 4 ml media. That should be sufficient for 20 slides.
- Dispense 200 μl of cell suspension onto a poly-L-Lysine slide.
- Place in 37c for ~ 4 hours, 5 slides per 100 mm dish.
- All steps from this point on are made at RT.
- Align two slides back-to-back, tilt to remove excess liquid and quickly dip in a PBS-filled coplin jar.
- Place 5 back-to-back slides (a total of 10) in a colplin jar filled with 40 ml of fixation buffer and fix for 10 minutes. Fixation buffer: 10 ml 16% ParaFormAldehyde (PFA, final conc. 4%), 4 ml 10X PBS and 26 ml UltraPure Water (UPW).
- Repeat step 5.
- Permeabilize for 10 minutes in a coplin jar. Permeabilization buffer: 0.5% Triton-X-100 (200 μl) in PBS. Store at cold room. Slides are stable for at least a couple of months.
- Picture attached.