How to prepare UF/UA stain solution (0.75%):
- Weigh out 37.5mg uranyl formate
- Heat 5mL water to a boil and then place into a tiny beaker with stir bar
- Add uranyl formate to beaker + water and stir for 5 min in the dark
- Add ~10uL 5N NaOH and stir for another 5 min in the dark. For this step, be very careful to watch for precipitation. The color should turn a bright yellow, but you don't want to see any precipitation. If you do, you've added too much NaOH. Scrap your solution and repeat steps 1-3, adding less NaOH at step 4.
- Filter solution through 0.5um filter (any size filter is probably fine - we just want to get big chunks out)
- Wait for it to cool before use. Hot stain may not work as intended.
- Keep the stain covered to avoid exposure to light.
- If you want to freeze tiny aliquots for later use, flash freeze (using LN2) in small eppendorf tubes and place in the -80
How to stain grids:
- Glow discharge your grids with the carbon face up (30 sec, ~15mA, exact numbers not super important)
- Apply 2-3uL of sample at 0.1-0.005mg/mL to carbon face of grid (good idea to do a range of concentrations if this is the first time looking at the sample).
- Let sample incubate on grid for 30 sec
- Blot off sample
- Apply buffer or water to carbon face of grid and aspirate off. Repeat a second time
- Apply stain solution to carbon face of grid and aspirate off.
- Apply stain solution to carbon face of grid a second time and wait 30 sec. Then aspirate off.
- Grids are now ready to image on the microscope. They may need a few minutes to dry before going into the column.