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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.
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Pelleting Cells for EM with 2.5% Glutaraldehyde in 0.1M Sodium Cacodylate Buffer Fixation

1. Cell Collection and Pelleting (suggested)

  • Harvest cells from culture by centrifugation:
    • Mammalian cells: 500鈥�1,000 脳 g for 5鈥�10 minutes at 4掳C.
    • Bacteria: 3,000鈥�10,000 脳 g for 10鈥�15 minutes at 4掳C.
  • Carefully remove the supernatant without disturbing the pellet.

2. Washing

  • Wash the pellet with ice-cold 1脳 PBS (0.1 碌m sterile-filtered) to remove media and secreted proteins.
  • Centrifuge again under the same conditions.
  • Repeat the wash once more.

3. Primary Fixation

  • Fix the washed pellet with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 30鈥�60 minutes at 4掳C (or longer, up to 72 hours, for extended fixation).
  • Avoid drying the sample during this step.
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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for听2 hours at 4鈥癈.
  5. Water Wash: Rinse tissue in听double-distilled water (ddH鈧侽), 3 times for 10 minutes each.
  6. Optional En Bloc Staining (If not required, skip to step 8):
    • For听elastin, microfilaments or microtubules: Incubate in听2% tannic acid in 0.1 M sodium cacodylate buffer听for听1 hour at 4鈥癈听on a tissue rotator.
    • For听FIB-SEM analysis: Stain with听2% aqueous uranyl acetate听for听1 hour.
  7. Water Wash: Rinse again in听ddH鈧侽, 3 times for 10 minutes each.
  8. Dehydration: Gradually dehydrate tissue in an听acetone:ddH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for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.

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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鈧勌齱ith 1 part ddH鈧侽听to make 2%.
    • Dissolve 0.3 g听potassium ferrocyanide in听10 mL听ddH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ddH鈧侽.
    • Protect it from light (wrap the container in foil).
    • Store at听4鈥癈.
    • Handle with care鈥攔adioactive and toxic.

5.听Acetone Series for Dehydration

  • Prepare fresh dilutions from 100% acetone using ddH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 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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