Microwave Tissue Processing Checklist Sample_______________________

Osmium-Based Chemistry for up to 6 micro-

centrifuge tubes, PELCO 3451 Microwave Oven Date _______________________

(MW), Power Controller, Cold Spot Device,

Vacuum Chamber, Load Cooler (set to 30 ^oC),

Ethanol Dehydration with 1% Uranyl Acetate

“en bloc” staining, Epon Resin

Aldehyde fixation step included

Standard Epon with Enhanced Ribosomal Staining

	Temperature Restriction Set Point	Processing Time in MW	Processing Time Outside MW at RT
1. Aldehyde Fixation--- UNDER vacuum Ø Microcentrifuge tubes Ø Cool samples below 15^oC. Power level = 100-175 W	37 ^oC 1X or 2X	__1 min ON__ __1 min OFF__ __1 min ON__ Pre-cool before each session.
2. 0.1 M Cacodylic buffer rinses			3-4X(can be stored overnight at RT)
3. Reduced Osmium--- UNDER vacuum Ø Mix vial 1 (1.5 ml of 4% OSO₄(aq) and 1.5 ml of 0.2 M Cacod) with vial 2 (1.5 ml of 6% KFeCN (aq) and 1.5 ml 0.2 M Cacod). Ø Cool samples below 15^oC. Power level = 100-175 W	37 ^oC 1X or 2X	__1 min ON__ __2 min OFF__ __1min ON__ Pre-cool before each session.
4. 0.1 M Cacodylic rinses			5X at 1-2 min each
5. 1% Osmium- UNDER vacuum Ø Mix 1.5 ml 4% OsO₄ (aq), 1.5 ml water and 3 ml of 0.2 M cacodylic buffer. Ø Cool samples below 15^oC. Power level = 100-175 W	37 ^oC 1X or 2X	__1 min ON__ __2 min OFF__ __1 min ON__ Pre-cool before each session.
6. 0.1 M Cacodylic rinses			5X at 1-2 min each
7. Water rinses			2X briefly
8. Ethanol dehydration with 1% uranyl acetate Do not use vacuum!!!!!! Ø Change to processing baskets. 50% Etoh 50% Etoh & 1% UA 70% Etoh & 1% UA 90% Etoh & 1% UA 100% Etoh & 1% UA 100% Etoh 1:1 Etoh to Acetone 1:2 Etoh to Acetone 100% Acetone 3X Power level @ 250 W	37 ^oC	40 sec___ 40 sec___ 40 sec___ 40 sec___ 40 sec___ 40 sec___ 40 sec___ 40 sec___ 40 sec__ __ __
9. Resin infiltration-UNDER vacuum Position Petri dishes in any area, EXCEPT in the lower right quadrant of the cold spot device. 1:1 Acetone to Epon 1:4 Acetone to Epon 100% Epon 3X Power level = 350 W	45 ^oC	3 min ___ 3 min ___ 3 min __ __ __
10. Polymerization for coffin molds or BEEM capsules Ø use fresh 100% Epon			60 ^oC oven, embed in molds, cure for 48 hours
11. Optional for coffin molds: Ø re-surface block with fresh Epon 24 to 36 hours into the 48 hour curing session			60 ^oC oven, embed on ACLAR, cure for at least 12 hours

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