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Sample Processing and Embedding for Electron Microscopy

Day 1 – Fixation, Post-Fixation, and Initial Dehydration

  1. Container: Perform all steps in clean glass vials to avoid plastic contamination.
  2. Primary Fixation: Use a fixation method appropriate for the tissue type (e.g., glutaraldehyde-based fixatives).
  3. Buffer Wash: Wash tissue in 0.1 M sodium cacodylate buffer, 4 times for 15 minutes each.
  4. Post-Fixation: Incubate tissue in 1% aqueous osmium tetroxide + 1.5% aqueous potassium ferrocyanide for 2 hours at 4 °C.
  5. Water Wash: Rinse tissue in double-distilled water (ddH₂O), 3 times for 10 minutes each.
  6. 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.
  7. Water Wash: Rinse again in ddH₂O, 3 times for 10 minutes each.
  8. 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)
  9. Infiltration (Start): Place tissue in 1:1 Epon:acetone mixture on a rotator overnight.

Day 2 – Resin Infiltration

  1. Infiltrate tissue in 2:1 Epon:acetone mixture on a rotator for 6–8 hours.
  2. Replace with 3:1 Epon:acetone mixture and continue rotating overnight.

Day 3 – Final Infiltration and Embedding

  1. Infiltrate tissue in 100% Epon on a rotator for 2 hours.
  2. Remove vial cap and place tissue under vacuum in 100% Epon for 2 hours to remove trapped air.
  3. 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.
  4. 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 g 23 g 34.5 g 46 g 57.5 g 69 g
DDSA 6.5 g 13 g 19.5 g 26 g 32.5 g 39 g
NMA 7.0 g 14 g 21 g 28 g 35 g 42 g
DMP-30 0.45 mL 0.9 mL 1.35 mL 1.8 mL 2.25 mL 2.7 mL

  • Dilutions:

    • Mix with acetone to make 1:1, 2:1, and 3:1 Epon:acetone solutions as needed.
    • Prepare fresh daily.
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