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Sample Processing and Embedding for Ultrastructural Morphology by Transmission Electron Microscopy

Adapted and Prepared by Jeannie Mui
 

Introduction

This protocol describes preparing biological tissues for ultrastructural analysis by transmission electron microscopy (TEM). The procedure includes post-fixation, optional en bloc staining, dehydration, resin infiltration, embedding, and polymerization using Epon epoxy resin.

General Considerations

  • Perform all reagent exchanges gently to minimize mechanical damage to the specimen.
  • Use clean glass vials throughout processing whenever possible to minimize contamination from plastics and solvent-induced leaching.
  • Ensure specimens remain fully submerged during all incubation steps.
  • Handle all hazardous reagents, including osmium tetroxide, uranyl acetate, acetone, and sodium cacodylate, in accordance with institutional safety regulations and within a certified chemical fume hood.
  • Processing times may require adjustment depending on specimen size, density, and fixation quality.
     

Tissue Processing

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

  1. Container: Perform all steps in clean glass vials to avoid plastic contamination.
  2. Primary Fixation: Fix tissue using an appropriate primary fixation protocol for the specimen type (typically aldehyde-based fixation, such as 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer).
  3. Buffer Wash: Wash tissue in 0.1 M sodium cacodylate buffer, 4 times for 15 minutes each.
  4. Reduced Osmium Post-Fixation: Incubate tissue in 1% aqueous osmium tetroxide + 1.5% aqueous potassium ferrocyanide for 2 hours at 4 °C.
    Important:
    • Ensure complete specimen immersion.
    • Protect reagents from prolonged light exposure.
    • Perform all handling in a fume hood.
  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):
     
    • Option A: Tannic Acid Treatment
      • For enhanced visualization of:
        • Elastin fibers
        • Microfilaments
        • Microtubules

        • Incubate in:

        • 2% tannic acid
        • Prepared in 0.1 M sodium cacodylate buffer for 1 hour at 4°C on a tissue rotator.
    • Option B: Uranyl Acetate Staining
      • For
        • FIB-SEM applications
        • Enhanced membrane contrast
        • Improved overall electron density

        • Incubate in:

        • 2% aqueous uranyl acetate for 1 hour at 4°C.
  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 as larger tissue pieces may require extended incubation times)
  9. Infiltration (Start): Place tissue in a 1:1 Epon: acetone mixture and rotate gently overnight.

Day 2 – Intermediate Resin Infiltration

  1. Incubate tissue in a 2:1 Epon: acetone mixture on a rotator for 6–8 hours.
  2. Replace with a 3:1 Epon: acetone mixture and rotate gently overnight.

Day 3 – Final Infiltration and Embedding

  1. Incubate tissue in 100% Epon for 2 hours with gentle rotation.
  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 moulds.
    • 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.

 

Monolayer Processing for Transmission Electron Microscopy (TEM)

Standard One-Day Processing

After primary fixation, process cell monolayers for TEM according to the procedure below. Perform all steps 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 incubation.
    • Perform this step in a chemical fume hood using appropriate personal protective equipment (PPE).

  3. Water Wash

    • Rinse monolayers in double-distilled water (ddH₂O) 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 hour at 4°C.

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

  5. Water Wash

    • Rinse samples again in ddH₂O 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.
    • Adjust dehydration times based on cell type, thickness, and degree of confluency.

  7. Resin Infiltration

    • Place samples on a slightly angled Belly Dancer shaker 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 resin to 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

  • Perform all reagent exchanges 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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