TITLE: Volume Electron Microscopy Protocol for the ASP-1000
AUTHORS: Erin S Stempinski, Jessica  Riesterer, Steven  Adamou, Claudia  S Lopez

[DESCRIPTION]
Volume electron microscopy (vEM) requires a multitude of heavy metal stains to ensure adequate sample contrast and conductivity under the electron beam. Bench protocols for vEM can be time- and labor-intensive. The mPrep™ ASP-1000™ automated specimen processor equipped with a thermal control unit reduces the amount of time and labor required for vEM protocols.


[STEPS]
SECTION: Prepare reagents and accessories
2. Prepare the reagents in the concentrations outlined in the materials section.

Freshly prepare day-of:
Osmium tetroxide (OsO4) solutions if from aqueous vials
1%  w/v thiocarbohydrazide (TCH), aqueous - requires 1 hour to dissolve at 60°C, swirling every 10 minutes.
EMBed812 resin

Can be prepared ahead:
0.1M sodium cacodylate
2.5% w/v potassium ferricyanide in 0.1M sodium cacodylate
1% w/v uranyl acetate, aqueous
Walton's lead stain, keep at 60°C
aqueous acetone, 50%, 75%, 85% 95%
SECTION: Set up ASP-1000
6. Place a reservoir in the Plate 1 slot and add 0.1M sodium cacodylate in each channel used.
SECTION: Add the protocol to the ASP-1000
1. Add the attached protocol to the ASP-1000 protocol list.   

SECTION: Set up ASP-1000
7. Under the fume hood, add reagents to a reservoir according to the plate map for Plate 2 and water for the rinse steps. Place a pierce heat seal foil plate sheet on top of the reservoir with the blue strip facing up. Run the heated iron across the foil starting from the middle of the tray and working to the edges.

Ensure that the iron has completely sealed the foil along the edges of the reservoir. Reservoir can be taken out of the fume hood and placed in the Plate 2 slot.
 

SECTION: Set up ASP-1000
5. Follow the attached plate map for placement of reagents in the ASP-1000 in the 12 channel reagent reservoirs.    Five to six milliliters of reagent will be placed in each channel, except for 100% acetone, which will require more.

SECTION: Prepare reagents and accessories
3. Heat the small iron to the maximum temperature.
SECTION: Set up ASP-1000
8. Ensure that Plate 3 has reached 60°C. Remove the magnetic top and place a reservoir in Plate 3. Add the fully-dissolved TCH to the appropriate area. Use water for the rinse steps. Replace the magnetic top.
SECTION: Prepare reagents and accessories
4. Heat the temperature controlled areas on the ASP-1000 to 60ºC using the thermal control unit software
SECTION: Set up ASP-1000
9. Ensure that Plate 4 has reached 60ºC. Remove the magnetic top and place a reservoir in Plate 4. Add Walton's lead stain to the appropriate area and use water for the rinse steps. Replace the magnetic top.
SECTION: Set up ASP-1000
10. Slide a reservoir into the Plate 6 slot. Add the uranyl acetate solution and use water for the rinse steps.
 

SECTION: Prepare the sample
11. 

 

Place a drop of 0.1M cacodylate buffer on a cutting surface.
Dissect the sample into 1mm3 pieces with a razor blade. Use a guillotine-like motion to minimize the damage of cells along the edge. 
SECTION: Prepare the sample
12. Place the mPrep/s capsules in the mPrep/Bench silicone rack. Place a few drops of 0.1M cacodylate buffer in a mPrep/s capsule. Place the samples in the mPrep/s capsule.
 

SECTION: Run the ASP-1000
13. Start the ASP-1000 protocol on the user interface. When prompted, attach the mPrep/s capsules to the pipette head. Remove excess fluid in the capsule with a Kimwipe or paper towel. Press "Enter" for the ASP-1000 to start the automated sample processing protocol.
SECTION: Run the ASP-1000
14. During the end of the uranyl acetate rinse steps or the beginning of the Walton's lead stain step, prepare the final reservoir for Plate 5 with the acetone series and resin infiltration.
 

 

Cover the reservoir with a pierce heat foil sheet with the blue strip up. As before, iron the foil sheet beginning from the middle and working outward. Slide the reservoir into the area for Plate 5.
SECTION: Run the ASP-1000
15. When prompted at the end of the automatic protocol, press "Enter" to raise the pipette head. Remove the capsules from the pipette head. 

SECTION: Run the ASP-1000
15.1. If polymerizing samples in the mPrep/s capsules, place the capsules in the mPrep/Bench silicone rack and add resin until desired height. Place the silicone rack in a 60°C oven.
SECTION: Run the ASP-1000
15.2. If polymerizing samples in other molds, remove the samples from the mPrep capsule and place in prepared mold. Add resin and polymerize in an oven at 60°C.
SECTION: Clean up
16. Dispose of reagents in accordance with local and institutional laws and regulations.

Remove the reagents from Plate 1 and Plate 6 and slide out the reservoirs. Reagents can removed from Plates 3 and 4 either before or after the reservoirs have been removed from the ASP-1000.

For Plate 2, place an x-pierce cross-cut plate seal sheet on top of the reservoir before removal. Place the reservoir in the fume hood before removing the reagents.

For Plate 5, slide the reservoir out of the ASP-1000. Remove any unevaporated acetone dilutions. Place the reservoir in a 60°C oven to polymerize.