Adapted and Prepared by Jeannie Mui
Processing usually takes three days, with an additional two days for polymerization.
Day 1 – Fixation, Post-Fixation, and Initial Dehydration
- Container: Perform all steps in clean glass vials to avoid plastic contamination.
- Primary Fixation: Use a fixation method appropriate for the tissue type (e.g., glutaraldehyde-based fixatives).
- Buffer Wash: Wash tissue in 0.1 M sodium cacodylate buffer, 4 times for 15 minutes each.
- Post-Fixation: Incubate tissue in 1% aqueous osmium tetroxide + 1.5% aqueous potassium ferrocyanide for 2 hours at 4 °C.
- Water Wash: Rinse tissue in double-distilled water (ddH₂O), 3 times for 10 minutes each.
- Optional En Bloc Staining (If not required, skip to step 8):
- For elastin, microfilaments or microtubules: Incubate in 2% tannic acid in 0.1 M sodium cacodylate buffer for 1 hour at 4 °C on a tissue rotator.
- For FIB-SEM analysis: Stain with 2% aqueous uranyl acetate for 1 hour.
- Water Wash: Rinse again in ddH₂O, 3 times for 10 minutes each.
- Dehydration: Gradually dehydrate tissue in an acetone:ddH₂O series:
- 30%, 50%, 70%, 80%, 90%, and 3 × 100% acetone
- 10 minutes per step (adjust based on tissue size)
- Infiltration (Start): Place tissue in 1:1 Epon:acetone mixture on a rotator overnight.
Day 2 – Resin Infiltration
- Infiltrate tissue in 2:1 Epon:acetone mixture on a rotator for 6–8 hours.
- Replace with 3:1 Epon:acetone mixture and continue rotating overnight.
Day 3 – Final Infiltration and Embedding
- Infiltrate tissue in 100% Epon on a rotator for 2 hours.
- Remove vial cap and place tissue under vacuum in 100% Epon for 2 hours to remove trapped air.
- Embedding:
- Transfer tissue to embedding molds.
- Add a paper label with the sample ID to one end of each mould at the opposite end of the tissue.
- Fill the mould with fresh Epon.
- Polymerization: Cure in an oven at 58–60 °C for 48 hours.
Reagent Preparation
1. 0.1 M Sodium Cacodylate Buffer
- Stock: Sodium cacodylate trihydrate
- Preparation:
- Dissolve 2.14 g of sodium cacodylate trihydrate in 100 mL of distilled water.
- Adjust pH to 7.2–7.4 with HCl.
- Store at 4 °C.
2. 1% Osmium Tetroxide + 1.5% Potassium Ferrocyanide (Post-Fixative)
- Stock: 4% aqueous osmium tetroxide (handle in fume hood)
- Preparation:
- Mix 1 part 4% OsO₄ with 1 part ddH₂O to make 2%.
- Dissolve 0.3 g potassium ferrocyanide in 10 mL ddH₂O to make 3% solution.
- Mix equal volumes of 2% OsO₄ and 3% potassium ferrocyanide immediately before use.
- Keep on ice during use.
3. 2% Tannic Acid in 0.1 M Sodium Cacodylate
- Preparation:
- Dissolve 0.2 g tannic acid in 10 mL of 0.1 M sodium cacodylate buffer.
- Filter before use.
- Store at 4 °C, use within a few days.
4. 2% Aqueous Uranyl Acetate (En Bloc Stain for FIB-SEM)
- Preparation:
- Dissolve 0.2 g uranyl acetate in 10 mL ddH₂O.
- Protect it from light (wrap the container in foil).
- Store at 4 °C.
- Handle with care—radioactive and toxic.
5. Acetone Series for Dehydration
- Prepare fresh dilutions from 100% acetone using ddH₂O:
- 30%, 50%, 70%, 80%, 90%, and 3 × 100%
- Use glassware to avoid plastic leaching.
6. Epon Resin Mixtures
-
Stock Components:
- Epon 812 resin
- DDSA (dodecenyl succinic anhydride)
- NMA (nadic methyl anhydride)
- DMP-30 (accelerator)
-
Typical Epon Mix:
Reagent | Amount for 25 mL | Amount for 50 mL | Amount for 75 mL | Amount for 100 mL | Amount for 125 mL | Amount for 150 mL |
---|---|---|---|---|---|---|
Epon | 11.5 | 23 | 34.5 | 46 | 57.5 | 69 |
DDSA | 6.5 | 13 | 19.5 | 26 | 32.5 | 39 |
NMA | 7.0 | 14 | 21 | 28 | 35 | 42 |
DMP-30 | 0.45 | 0.9 | 1.35 | 1.8 | 2.25 | 2.7 |
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Dilutions:
- Mix with acetone to make 1:1, 2:1, and 3:1 Epon:acetone solutions as needed.
- Prepare fresh daily.