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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 鈥� Fixation, Post-Fixation, and Initial Dehydration

  1. Container: Perform all steps in clean听glass vials听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听0.1 M sodium cacodylate buffer, 4 times for 15 minutes each.
  4. Post-Fixation: Incubate tissue in听1% aqueous osmium tetroxide + 1.5% aqueous potassium ferrocyanide听蹿辞谤听2 hours at 4鈥癈.
  5. Water Wash: Rinse tissue in听double-distilled water (诲诲贬鈧侽), 3 times for 10 minutes each.
  6. Optional En Bloc Staining (If not required, skip to step 8):
    • 贵辞谤听elastin, microfilaments or microtubules: Incubate in听2% tannic acid in 0.1 M sodium cacodylate buffer听蹿辞谤听1 hour at 4鈥癈听on a tissue rotator.
    • 贵辞谤听FIB-SEM analysis: Stain with听2% aqueous uranyl acetate听蹿辞谤听1 hour.
  7. Water Wash: Rinse again in听诲诲贬鈧侽, 3 times for 10 minutes each.
  8. Dehydration: Gradually dehydrate tissue in an听acetone:诲诲贬鈧侽 series:
    • 30%, 50%, 70%, 80%, 90%, and 3 脳 100% acetone
    • 10 minutes per step (adjust based on tissue size)
  9. Infiltration (Start): Place tissue in听1:1 Epon:acetone听mixture on a rotator听overnight.

Day 2 鈥� Resin Infiltration

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

Day 3 鈥� Final Infiltration and Embedding

  1. Infiltrate tissue in听100% Epon听on a rotator for听2 hours.
  2. Remove vial cap and place tissue under听vacuum in 100% Epon听蹿辞谤听2 hours听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听58鈥�60鈥癈 for 48 hours.

听

Reagent Preparation

1.听0.1 M Sodium Cacodylate Buffer

  • Stock: Sodium cacodylate trihydrate
  • Preparation:
    • Dissolve听2.14 g听of sodium cacodylate trihydrate in听100 mL听of distilled water.
    • Adjust pH to听7.2鈥�7.4听with HCl.
    • Store at听4鈥癈.

2.听1% Osmium Tetroxide + 1.5% Potassium Ferrocyanide (Post-Fixative)

  • Stock: 4% aqueous osmium tetroxide (handle in fume hood)
  • Preparation:
    • Mix听1 part 4% OsO鈧�听with 1 part 诲诲贬鈧侽听to make 2%.
    • Dissolve 0.3 g听potassium ferrocyanide in听10 mL听诲诲贬鈧侽 to make 3% solution.
    • Mix equal volumes of 2% OsO鈧� and 3% potassium ferrocyanide听immediately before use.
    • Keep on听ice听during use.

3.听2% Tannic Acid in 0.1 M Sodium Cacodylate

  • Preparation:
    • Dissolve 0.2 g听tannic acid in听10 mL听of 0.1 M sodium cacodylate buffer.
    • Filter before use.
    • Store at听4鈥癈, use within a few days.

4.听2% Aqueous Uranyl Acetate (En Bloc Stain for FIB-SEM)

  • Preparation:
    • Dissolve 0.2 g听uranyl acetate in听10 mL听诲诲贬鈧侽.
    • Protect it from light (wrap the container in foil).
    • Store at听4鈥癈.
    • Handle with care鈥�radioactive and toxic.

5.听Acetone Series for Dehydration

  • Prepare fresh dilutions from 100% acetone using 诲诲贬鈧侽:
    • 30%, 50%, 70%, 80%, 90%, and 3 脳 100%
    • Use听glassware听to avoid plastic leaching.

6.听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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