Longhand

Epon:

• Place stir bars in two tri-pour beakers and label A and B.
• Measure resin and hardeners. These amounts vary according to the WPE of the resin, so update as needed using the formula from Ladd. For a WPE of 144:
  • Into beaker A measure 7.00g LX112 and 8.99g DDSA.
  • Into beaker B measure 18.00g LX112 and 15.47g NMA.
• Cover both beakers with aluminum foil and place on stir plates.
• Beakers should mix for at least 15 minutes. Do not mix too vigorously to avoid bubbles, just enough to have a slight vortex.
• Into a third tri-pour beaker, pour 9g of beaker A and 21g of beaker B.
• Mix for another 15 minutes.
• Add 0.42g of DMP-30 and mix for another 10-15 minutes.

Label:

• Tripour beakers for UA waste and osmium waste.
• 6 2mL vials: A,B,C,D,E,F
• 18 scintillation vials
  • 2×(50,70,90,100) for ETOH/UA
  • Osm
  • 1
  • 2
  • KFeCN
  • 50% ETOH
  • 1:1 ETOH:propylene oxide
  • 1:2 ETOH:propylene oxide
  • 100% propylene oxide
  • 1:1 propylene oxide:epon with DMP-30
  • 1:2 propylene oxide:epon with DMP-30

Set out:

• A 50mL falcon tube filled with water
• A 50mL falcon tube filled with 0.1M cacodylate
• A 20cc scintillation vial filled with 0.2M cacodylate
• 10 60mm polypropylene processing dishes
• 4 20mL syringes and 4 acro disc syringe filter tips (0.2 µ m pore size)
• Solid waste bags for UA and toxic waste

Solvents:

• Make vial of 50% ETOH in water.
• Mix vials of 1:1 and 1:2 ETOH to acetone.

Osmium:

• To the scintillation vial labeled KFeCN add 0.3g KFeCN and 5mL water. Mix well by shaking.
• Make vials of reduced and regular osmium:
  • Add 3mL 0.2M cacodylate buffer to vials 1 and 2 and 6mL to vial OS.
  • Add 3mL 4% OsO 4 to vial 1 and vial OS.
  • Add 3mL 6% KFeCN to vial 2.
  • Add 3mL water to OS vial.
• Place vials on ice.
• When ready, pour vials 1 and 2 back and forth to mix before adding to tissue.

Uranyl Acetate:

• Add 5mL 2% ethanolic UA solution (1g UA in 50mL EtOH) to one set of four dehydration vials.
• Add water and ETOH into vials to total 10mL each (5:0mL, 3:2, 1:4, 0:5 respectively for 50, 70, 90, and 100%) to end with 1%UA.
• Cap vials and shake to mix.
• Filter each UA vial through a syringe with a filter tip into a new, clean scintillation vial.

Microwave preparation:

• Fill load cooler with fresh deionized water and attach tubing to cold spot device. Make sure pump is on.
• Set microwave power level to 1 (175W).
• Run microwave for 2 minutes to warm up magnetron.

Tissue transfer and rinse:

• Fill one third of a processing dish with 0.1M cacodylate buffer.
• Transfer tissue to processing basket with a pipet.
• Transfer basket to another dish filled with cacodylate.
• Rinse 4 more times with cacodylate buffer.

Osmication:

• Pour vial 1 into vial 2, and continue to pour between the two until they are well mixed.
• Pour reduced osmium mixture into a processing dish and place basket in dish.
• Leave basket in dish for 5 minutes.
• Rinse 5 times with 0.1M cacodylate.
• Fill a dish with 1% osmium and transfer the basket.
• Microwave under vacuum 1' on/1' off/ 1' on
• Place dish on ice, cool to below 15° and repeat.

Rinse:

• Rinse 5 times with 0.1M cacodylate, waiting at least 2 minutes each time.
• Rinse 2 times with water.
• Replace water with 50% ETOH.

Dehydration:

• Turn microwave to power level 2 (250W)
• Transfer basket with into dishes of the following and microwave 40s without vacuum :
  • 50% ETOH/UA
  • 70% ETOH/UA
  • 90% ETOH/UA
  • 100% ETOH/UA
  • 100% ETOH

END MICROWAVE PROCESSING AND SWITCH TO LONG-HAND

Infiltration:

• Transfer tissue from basket into labeled vials filled with 1:1 EtOH:propylene oxide for 10 minutes on rotator
• 1:2 EtOH:propylene oxide for 10 minutes on rotator
• 100% propylene oxide for 2x15 minutes on rotator
• 1:1 propylene oxide:epon with DMP-30 for 1 hour on rotator
• 1:2 propylene oxide:epon with DMP-30 overnight on rotator

Embedding (the next day):

• Make fresh epon with DMP-30
• 3x1 hour in 100% epon on rotator
• Place labels writing side up in deep coffin molds and cover with fresh epon.
• Bevel a wooden applicator stick with a razor blade. Using the stick, transfer tissue into molds.
• Under the stereomicroscope, move the tissue so that it is at the bottom of the mold with the cell body layer away from the edge and parallel to it.
• Make sure that the epon is level with the top of the mold.
• Place mold in 60° oven for 48 hours.

Waste containers:

• In addition to solid waste bags for UA and other toxic waste, there should be labeled bottles for the following:
  • aldehydes, ethanol, acetone, and cacodylate
  • epon, ethanol, and acetone
  • osmium, KFeCN, and cacodylate
  • uranyl acetate
• If no waste container exists, make one.
• Pour all liquid waste into the proper container.
• Gloves, pipets, and lab paper go in solid waste bags.

Glassware and equipment:

• Beaker used for UA waste should be rinsed into the UA waste bottle and disposed of in the solid UA waste bag.
• Tri-pour beakers used for resins should be rinsed well with acetone into the waste bottle and left in the hood to dry.
• Stir bars and processing baskets should be placed in sealed specimen cups of acetone and left in the hood for several days to soak, changing the solvent daily.
• One resin beaker should be used to collect all excess resin and plastic pipets and dishes used with it. This should be cured in the oven with the blocks and discarded in toxic waste.
• Monitor the area for radioactivity.