Volume Electron Microscopy Protocol for the ASP-1000

Add the attached protocol to the ASP-1000 protocol list. ASP1000_protocol.pdf

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%

Heat the small iron to the maximum temperature.

Heat the temperature controlled areas on the ASP-1000 to 60ºC using the thermal control unit software

Follow the attached plate map for placement of reagents in the ASP-1000 in the 12 channel reagent reservoirs. ASP1000_platemap.pdf Five to six milliliters of reagent will be placed in each channel, except for 100% acetone, which will require more.

Place a reservoir in the Plate 1 slot and add 0.1M sodium cacodylate in each channel used.

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.

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.

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.

Slide a reservoir into the Plate 6 slot. Add the uranyl acetate solution and use water for the rinse steps.

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.

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.

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.

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.

When prompted at the end of the automatic protocol, press "Enter" to raise the pipette head. Remove the capsules from the pipette head.

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.

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.

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.