McGill.CA / Facility for Electron Microscopy Research / Resources / Protocols

Routine Tissue Processing for Electron Microscopy

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

  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 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

  • 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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