Synaptek Grid Stick Kit
EMS Catalog #71175
Uranyl Acetate - Lead Citrate Staining with SynapTek Grid Staining Kit
Preparing 10% Uranyl Acetate
- 10 g Uranyl Acetate (EMS #22400)
- 100 ml Methanol (EMS #18510)
Place solution in a glass bottle with ground glass stopper (EMS 64202-A) and sonicate or shake until totally dissolved. You may want to filter or spin down, but we find it is usually not necessary. Store the solution in a dark bottle in freezer or refrigerator, but use solution at room temperature.
- Three methanol washes of 5 - 10 ml each.
0.1 % Lead Citrate
- .1 g Lead Citrate ( EMS #17800)
- 100 ml freshly distilled water
(See tech note in EMS catalog "Preparation of lead stains and avoiding precipitation).
Add 1 ml drop by drop of 10 N NaOH (0.4g NaOH per 1 ml) after lead has dissolved in water until the solution is clear. Store in 10 ml syringes or any airtight bottle at room temperature.
- NaOH pellets may be inserted into the rubber bulb to insure a carbon dioxide free pipette during lead staining.
0.1N NaOH Wash
- Make 10 ml 0.1N NaOH wash solution and adjusted pH to 12 using HCl
100 ml of Freshly Distilled Water
- Use a narrow mouth stoppered bottle rather than beaker to store the water.
Place up to ten grids on the stick. If you are using formvar-coated grids, be sure formvar is away from the GridStick's surface and the slot runs in the same direction as the Grid Stick's slot. It helps to place the stick on a clean, flat surface and to push down with forceps on the outer edge of the grid against the adhesive to insure good contact. If you are using thin mesh grids, it is only necessary to lightly push on the outer rim. Although the pipettes have been treated to remove all organic residues as well as washed and blown dry with filtered air, it is always a good practice to rinse the pipette with a few methanol washes before starting.
Place the GridStick™ inside the first pipette and insert the rubber bulb with red plug in the pipette's top. Push the bulb's rubber rim down around the pipette to prevent accidental release. The red plug has a very small hole on its side and serves two important functions. First, it prevents any dust particles from entering the pipette, and second, it limits the rate of flow of staining and washing solutions, thereby limiting pressure changes so that formvar films do not break. Gently aspirate one to two milliliters of uranyl acetate into the pipette so that all grids are fully immersed and place the tip in an empty beaker. DO NOT leave pipette in uranyl acetate beaker or the solution will be aspirated into the rubber bulb. Place in the dark or subdued light for 10 - 30 minutes.
Dispose of uranyl acetate solution and rinse at least three times in separate methanol washes.
Remove GridStick™ from pipette One and place in a petri dish or some other dust-free area to dry about 30 minutes. You can leave it in the pipette, but drying time will increase to about one hours.
After grids are totally dry (very important that they are dry) place the SnapTek Grid Stick in pipette Two and gently aspirate one to two milliliters of the NaOH wash solution so that all grids have been immersed at least once. Since water has a high surface tension it is important that all aqueous solution be aspirated slowly. If you become too cavalier in this step, you may find small hole in your formvar films
The NaOH wash will purge the pipette of CO 2 and is usually more than adequate, but if you like, you may also place a few pellets of NaOH in the second rubber bulb, and pump it several times. No CO 2 will remain in the pipette. Gently aspirate the lead staining solution in the just-washed pipette and stain for three minutes.
Wash one again in the 0.1N NaOH
Wash the grids in a 100mcc beaker of distilled water at least dozen times. Be careful and go slowly. One effective wash method is to remove the rubber bulb and simply dip the entire pipette with GridStick™ inside, into the beaker. Lift the pipette out of the beaker and let the water drain, and so on for a dozen or more times
Remove SynapTek™ GridStick™ from pipette and let grids dry.
Remove Grid Stick and place in a pipette with red caps for storage. Grids may be stored for long periods of time in a tube. Our experience has been that they are safe for months and we think they will be secure for years and most likely decades.
When ready to examine grids, place fine forceps in undercut notch of the Grid Stick and remove grids. You may reuse the GridStick™ at least six or more times