Grid-Stick Kit

EMS Catalog #71175

This Technical Data Sheet describes the protocol for Synaptek GridStick staining with Uranyl Acetate - Lead Citrate.

The Grid-Stick Kit contains the following

• Coated Grid Sticks (5)
• Staining Pipettes (modified) (10)
• Flow-limiting plugs and bulbs (2)

Application of the GridStick

• Point drying of whole mounts
• Collection of serial sections
• Preliminary inspection of biological material at the light level using a microscope
• Special staining procedures
• Preliminary check of Formvar films on slot grids at the light level
• Grid support for carbon coating
• Emulsion coating for autoradiography

Instructions

10% Uranyl Acetate (10gm Uranyl Acetate; 100 ml Methanol)

1. Place the solution into a glass flask with a cap, and shake thoroughly until the solution is completely dissolved.

Note: Though not required or always necessary, you may choose to filter the solution.

2. Store in a freezer or refrigerator with no light.

100% Methanol

1. Three (3) Methanol washes, each 5-10 ml.

0.1% Lead Citrate

• 0.1 gm Lead Citrate
• 100 ml freshly distilled water

1. Add 1 ml drop of 10N NaOH (0.4gm NaOH per 1 ml)

Note: Prior to this step, make sure that the lead has dissolved in water and that the solution appears clear.

2. Store in a container that is airtight at room temperature.

NaOH Pellets

Note: NaOH pellets can be placed into the rubber bulb. Doing so avoids carbon dioxide pipettes during the lead staining process.

0.1N NaOH Wash

1. Make a 10ml 0.1N NaOH wash solution and adjust the pH to 12 using HCL.

100cc of freshly distilled water
Clean glass beaker

Procedure

1. Place up to 10 grids on the stick.

Note: If you are using Formvar-coated grids in your application, be sure that the Formvar is of significant distance from the surface of the GridStick. Confirm that the slot runs in the same direction as the GridStick's slot. By following this procedure, the placement of the stick becomes better facilitated, as it is on a clean surface.

If your application calls for mesh grids, you only need to push on the outer rim, whereas opposed to the Formvar grids, where pushing is exerted onto the outer edge of the grid. We highly recommend rinsing the pipette with a few methanol washes prior to use, simply due to the high risk of residues and contaminants remaining.

2. Place the GridStick inside the first pipette and insert the rubber bulb containing the red plug in the top of the pipette. The very tiny hole on the side of the red plug serves two purposes: (1) to prevent contaminants from entering the pipette; and (2) to limit the rate of flow of staining and washing solutions.
3. Carefully take one to two cc's of Uranyl Acetate solution to the pipette, ensuring the full immersion of all grids, and place the tip into an empty beaker.

Note: Do not leave the pipette in the Uranyl Acetate or else it will result in the rubber bulb being aspirated.

4. Place in a dark area or wherever there is a reduced amount of light for 10-30 minutes.
5. Any remaining Uranyl Acetate should be disposed and rinsed at least three times in SEPARATE methanol washes.
6. Remove the GridStick from the first pipette and place it into a petri dish. Let dry for approximately 30 minutes.

Note: Should you choose to leave the GridStick in the pipette, please note that drying time will increase to about one hour.

7. One the grids are COMPLETELY dry, place the Synaptek GridStick into the second pipette and carefully aspirate one to two cc's of the NaOH wash solution, ensuring that all grids have been immersed at least once in the solution.

Note: Due to the high level of surface tension of the water, be sure that all aqueous solutions are aspirated in a slow, gradual manner. Failure to follow this step will result in the formation of small holes in your Formvar films.

8. At this point, the NaOH wash solution will purge the pipette of CO₂. If you prefer, you may choose to place a few pellets of NaOH into the second rubber bulb and pump it several times. No CO₂ will remain in the pipette. Gently aspirate the lead staining solution in the pipette that has just been washed, and stain for 3 minutes
9. Wash once more in the 0.1N NaOH.
10. Now, wash the grids in a 100 cc beaker of distilled water at least 12 times, slowly. A recommended and effective method for washing is to remove the rubber bulb and dip the entire pipette with the GridStick inside, into the beaker of distilled water.
11. Remove the pipette from the beaker and let the water drain, doing so several times.
12. Remove the Synaptek GridStick from the pipette and allow the grids sufficient time to dry.
13. Remove the GridStick and place it into a pipette with red caps and store. Grids can be stored for long period of time in a tube – in majority of cases, they have been efficiently stored for months, and have the potential to be stored up to years.
14. When the user is ready to being the examination of the grids, place the fine forceps in the undercut notch section of the GridStick and begin to remove the grids. The GridStick can be reused up to 6 times.

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Grid Stick Kit