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Sample Processing and Embedding for Morphology by Electron Microscopy

Adapted and Prepared by Jeannie Mui
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Tissue

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

  1. Container: Perform all steps in cleanÌýglass vialsÌýto 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 inÌý0.1 M sodium cacodylate buffer, 4 times for 15 minutes each.
  4. Post-Fixation: Incubate tissue inÌý1% aqueous osmium tetroxide + 1.5% aqueous potassium ferrocyanideÌýforÌý2 hours at 4 °C.
  5. Water Wash: Rinse tissue inÌýdouble-distilled waterÌý(ddHâ‚‚O), 3 times for 10 minutes each.
  6. Optional En Bloc StainingÌý(If not required, skip to step 8):
    • ForÌýelastin, microfilaments or microtubules: Incubate inÌý2% tannic acid in 0.1 M sodium cacodylate bufferÌýforÌý1 hour at 4 °CÌýon a tissue rotator.
    • ForÌýFIB-SEM analysis: Stain withÌý2% aqueous uranyl acetateÌýforÌý1 hour.
  7. Water Wash: Rinse again inÌýddHâ‚‚O, 3 times for 10 minutes each.
  8. Dehydration: Gradually dehydrate tissue in anÌýacetone:ddHâ‚‚O series:
    • 30%, 50%, 70%, 80%, 90%, and 3 × 100% acetone
    • 10 minutes per step (adjust based on tissue size)
  9. Infiltration (Start): Place tissue inÌý1:1 Epon:acetoneÌýmixture on a rotatorÌýovernight.

Day 2 – Resin Infiltration

  1. Infiltrate tissue inÌý2:1 Epon:acetoneÌýmixture on a rotator forÌý6–8 hours.
  2. Replace withÌý3:1 Epon:acetoneÌýmixture and continue rotatingÌýovernight.

Day 3 – Final Infiltration and Embedding

  1. Infiltrate tissue inÌý100% EponÌýon a rotator forÌý2 hours.
  2. Remove vial cap and place tissue underÌývacuum in 100% EponÌýforÌý2 hoursÌýto 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 atÌý58–60 °C for 48 hours.

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Reagent Preparation

1.Ìý0.1 M Sodium Cacodylate Buffer

  • Stock: Sodium cacodylate trihydrate
  • Preparation:
    • DissolveÌý2.14 gÌýof sodium cacodylate trihydrate inÌý100 mLÌýof distilled water.
    • Adjust pH toÌý7.2–7.4Ìýwith HCl.
    • Store atÌý4 °C.

2.Ìý1% Osmium Tetroxide + 1.5% Potassium Ferrocyanide (Post-Fixative)

  • Stock: 4% aqueous osmium tetroxide (handle in fume hood)
  • Preparation:
    • MixÌý1 part 4% OsOâ‚„Ìýwith 1-part ddHâ‚‚OÌýto make 2%.
    • Dissolve 0.3 gÌýpotassium ferrocyanide inÌý10 mLÌýddHâ‚‚O to make 3% solution.
    • Mix equal volumes of 2% OsOâ‚„ and 3% potassium ferrocyanideÌýimmediately before use.
    • Keep onÌýiceÌýduring use.

3.Ìý2% Tannic Acid in 0.1 M Sodium Cacodylate

  • Preparation:
    • Dissolve 0.2 gÌýtannic acid inÌý10 mLÌýof 0.1 M sodium cacodylate buffer.
    • Filter before use.
    • Store atÌý4 °C, use within a few days.

4.Ìý2% Aqueous Uranyl Acetate (En Bloc Stain for FIB-SEM)

  • Preparation:
    • Dissolve 0.2 gÌýuranyl acetate inÌý10 mLÌýddHâ‚‚O.
    • Protect it from light (wrap the container in foil).
    • Store atÌý4 °C.
    • Handle with care—radioactive and toxic.

5.ÌýAcetone Series for Dehydration

  • Prepare fresh dilutions from 100% acetone using ddHâ‚‚O:
    • 30%, 50%, 70%, 80%, 90%, and 3 × 100%
    • UseÌýglasswareÌýto avoid plastic leaching.

6.ÌýEpon 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.

Ìý

Monolayer Processing for Transmission Electron Microscopy (TEM)

Standard One-Day Processing

Following primary fixation, cell monolayers are processed for TEM according to the procedure below. All steps should be performed gently to avoid disruption or detachment of the cell layer.

  1. Buffer Wash

    • Wash samples in 0.1 M sodium cacodylate buffer three times for 15 minutes each at room temperature.
    • Alternatively, samples may remain in washing buffer overnight at 4°C if processing is delayed.

  2. Post-Fixation

    • Incubate samples in 1% aqueous osmium tetroxide containing 1.5% aqueous potassium ferrocyanide for 1 hour at 4°C.
    • Ensure samples are fully covered with fixative throughout the incubation period.
    • Perform this step in a chemical fume hood using appropriate personal protective equipment (PPE).

  3. Water Wash

    • Rinse monolayers in double-distilled water (ddHâ‚‚O) three times for 10 minutes each to remove residual osmium and buffer salts.

  4. Optional En Bloc Staining

    Depending on the experimental objectives, one of the following staining procedures may be applied:

    • For enhanced visualization of elastin, microfilaments, and microtubules:

      • Incubate in 2% tannic acid prepared in 0.1 M sodium cacodylate buffer for 1 hour at 4°C.

    • For FIB-SEM or enhanced membrane contrast:

      • Incubate in 2% aqueous uranyl acetate for 1 hourÌý4°C.

    If en bloc staining is not required, proceed directly to Step 6.

  5. Water Wash

    • Rinse samples again in ddHâ‚‚O three times for 15 minutes each to remove excess staining reagents.

  6. Dehydration

    • Dehydrate the monolayers through a graded ethanol series:
      • 30% ethanol
      • 50% ethanol
      • 70% ethanol
      • 80% ethanol
      • 90% ethanol
      • 100% ethanol (3 changes)
    • Incubate for approximately 8 minutes per step.
    • Dehydration times may be adjusted depending on cell type, thickness, and degree of confluency.

  7. Resin Infiltration

    • Place samples on a slightly angled Belly DancerÌýshaker or equivalent low-speed orbital mixer. Ensure containers are stable and solutions do not spill during agitation.

    Infiltration Schedule:

    • 1:1 Epon:Ethanol for 1 hour
    • 3:1 Epon:Ethanol for 1 hour
    • 100% Epon for 1 hour

  8. Embedding

    • Replace with fresh 100% Epon resinÌýto the top of each well, minimizing air bubbles.

  9. Polymerization

    • Cure the embedded samples in a 65°C oven for 48 hours or until the resin has fully polymerized.

Notes

  • All reagent exchanges should be performed carefully to prevent detachment of the cell monolayer.
  • Osmium tetroxide, uranyl acetate, and sodium cacodylate are hazardous chemicals and must be handled according to institutional safety guidelines.
  • Record all deviations from the protocol, including modified staining or dehydration times, as these may influence ultrastructural preservation and image quality.
  • Following polymerization, resin blocks are ready for block trimming, ultramicrotomy, and TEM examination.
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