Temperature Restriction Set Point

Processing Time in Microwave

Processing Time Outside Microwave at Room Temp.

1. 0.1 M Cacodylic Buffer Rinses

3-4x (can be stored overnight at RT)

2. Reduced Os - UNDER vacuum

Ø       Mix vial 1 (1.5 mls of 4% OsO₄ (aq) and 1.5 mls of 0.2 M cacod.) with vial 2 (1.5 mls of 6% KFeCN (aq) and 1.5 mls of 0.2M cacod.).

Ø       Microcentrifuge tubes

Ø       Cool samples below 15^o C

Power level = 100-175 W

37^o C

1X or 2X

__1 min ON,__

__2 min OFF,__

__1 min ON__

Pre-cool before each session.

3. 0.1 M Cacodylic Buffer Rinses

5x at 1-2 min each

4. 1% Osmium - UNDER vacuum

Ø       Mix 1.5 mls of 4% OsO₄ (aq), 1.5 mls water, and 3 mls of 0.2M cacod.

Ø       Cool samples below 15^o C.

Power level = 100-175 W

37^o C

1X or 2X

__1 min ON,__

__2 min OFF,__

__1 min ON__

Pre-cool before each session.

5. 0.1 M Cacodylic Buffer Rinses

5x at 1-2 min each

6. Water Rinses

2x briefly

7. 1% Uranyl Acetate (aq) – UNDER vacuum

Ø       Cool samples below 15^o C.

Power level = 100-175 W

37^o C

1X or 2X

__1 min ON,__

__2 min OFF,__

__1 min ON__

Pre-cool before each session.

8. Water Rinse

2x briefly

9. Acetone Dehydration

Do Not Use Vacuum!!!

Ø       Change to processing baskets

·   50% acetone     2X

·   70% acetone

·   90% acetone

·   100% acetone   3X

    Power level = 250 W

37^o C

40 sec __ __

40 sec __

40 sec __

40 sec __ __ __

10. Resin Infiltration – UNDER vacuum

Ø       1:1 Acetone to Epon/Spurr’s

Ø       100% Epon/Spurr’s 3X

Power level = 350 W

45^o C

3 min __

3 min __ __ __

For coffin molds or BEEM capsules:

11. Polymerization

Ø       use fresh 100% Epon/Spurr’s

60^o C oven, embed in molds, cure for 48 hours

12. Optional for coffin molds:

Ø Re-surface block with fresh Epon on Aclar at 24 to 36 hours into the 48 hour 60^oC curing session.

60^o C oven, embed on Aclar, cure for at least 12 hours